Anti-laminin4 antibodies specific for lg4-5

ABSTRACT

The invention provides antibodies that specifically bind to the LG4-5 modules of the G domain of laminin α4. The antibodies can preferentially stain cancer or tumor cells or tissue. The antibodies can be used for detecting cancer, evaluating the efficacy of a cancer therapy, treating cancer, and treating obesity or obesity-related diseases, among other applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 14/656,619 filed Mar.12, 2015, which claims the benefit of U.S. 61/952,132, filed Mar. 12,2014, U.S. 62/023,760 filed Jul. 11, 2014, and U.S. 62/068,349 filedOct. 24, 2014, each of which is incorporated herein by reference in itsentirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

The Sequence Listing written in file 510136SEQLST.txt is 144,516 bytes,was created on Mar. 12, 2018, and is hereby incorporated by reference.

BACKGROUND

Cancer is a major public health problem. One in four deaths in theUnited States is due to cancer, and the lifetime probability of beingdiagnosed with an invasive cancer is approximately 40%. Siegel et al.,CA Cancer J. Clin. 62:10-29 (2012). An estimated 500,000 Americans diefrom cancer each year (Siegel et al., CA Cancer J. Clin. 62:10-29(2012)). Lack of specificity of conventional chemotherapies andconsequent side effects limit the delivery of drug dosages needed toeliminate the majority of cancer cells.

Laminin proteins containing the laminin α4 chain have been reported tobe expressed in some types of cancer. Syndecans, which are reportedbinding partners of laminin α4, have also been reported to be expressedin some types of cancer.

SUMMARY OF THE CLAIMED INVENTION

The invention provides antibodies that specifically binds to an epitopewithin the LG4-5modules of the G domain of laminin α4. Some antibodiesdo not inhibit binding of laminin α4 to MCAM. Some antibodies bind to anepitope within LG4. Some antibodies bind to an epitope within LG5. Someantibodies bind to an epitope to which both LG4 and LG4 contributeresidues. Some antibodies inhibit binding of laminin α4 to a heparansulfate proteoglycan, such as syndecan-1, -2, -3, -4, a glypican, abetaglycan, CD44, perlecan, agrin, or collagen XVIII.

Some of the above antibodies selectively binds to a biological samplecomprising cancer cells, such as melanoma cells, when compared to acontrol sample, optionally wherein the control sample and the biologicalsample comprise cells of the same tissue origin and optionally whereinbinding of the antibody to the biological sample is at least 2-fold or5-fold greater than the binding of the antibody to the control sample.

Some of the above antibodies compete for binding to laminin α4 withantibody 15F7 characterized by a mature heavy chain variable region ofSEQ ID NO:16 and mature light chain variable region of SEQ ID NO:17, orantibody 6C12 characterized by a mature heavy chain variable region ofSEQ ID NO:26 and mature light chain variable region of SEQ ID NO:27, orantibody 13G10 characterized by a mature heavy chain variable region ofSEQ ID NOS:36 or 37 and mature light chain variable region of SEQ IDNO:38. Some of the above antibodies compete for binding to laminin α4with antibody 15F7 characterized by a mature heavy chain variable regionof SEQ ID NO:16 and mature light chain variable region of SEQ ID NO:17,or antibody 6C12 characterized by a mature heavy chain variable regionof SEQ ID NO:26 and mature light chain variable region of SEQ ID NO:27or 94, or antibody 13G10 characterized by a mature heavy chain variableregion of SEQ ID NOS:36 or 37 and mature light chain variable region ofSEQ ID NO:38.

Some of the above antibodies binds to the same epitope on laminin α4 as15F7, 6C12, or 13G10.

Some of the above antibodies comprising three light chain CDRs and threeheavy chain CDRs, wherein each CDR has at least 90% sequence identity toa corresponding CDR from the heavy and light chain variable regions of15F7 (SEQ ID NOS:16 and 17, respectively), 6C12 (SEQ ID NOS:26 and 27,respectively), or 13G10 (SEQ ID NOS:36/37 and 38, respectively). Some ofthe above antibodies comprising three light chain CDRs and three heavychain CDRs, wherein each CDR has at least 90% sequence identity to acorresponding CDR from the heavy and light chain variable regions of15F7 (SEQ ID NOS:16 and 17, respectively), 6C12 (SEQ ID NOS:26 and27/94, respectively), or 13G10 (SEQ ID NOS:36/37 and 38, respectively).

Some of the above antibodies comprise three heavy chain CDRs and threelight chain CDRs of 15F7, 6C12, or 13G10.

Some of the above antibodies are monoclonal. Some of the aboveantibodies are chimeric, humanized, veneered, or human. Some of theabove antibodies have human IgG1 kappa isotype.

The invention further provides a humanized or chimeric 15F7 antibodythat specifically binds to laminin α4, wherein 15F7 is a mouse antibodycharacterized by a mature heavy chain variable region of SEQ ID NO:16and a mature light chain variable region of SEQ ID NO:17. Optionally thehumanized antibody comprises a humanized heavy chain comprising threeCDRs of the 15F7 heavy chain variable region (SEQ ID NO:16) and ahumanized light chain comprising three CDRs of the 15F7 light chainvariable region (SEQ ID NO:17). Optionally the humanized antibodycomprises a humanized mature heavy chain variable region having an aminoacid sequence at least 90% identical to SEQ ID NO:58 and a humanizedmature light chain variable region having an amino acid sequence atleast 90% identical to SEQ ID NO:59. Optionally, the humanized antibodycomprises a humanized heavy chain comprising three CDRs of the 15F7heavy chain variable region (SEQ ID NO:16) and a humanized light chaincomprising three CDRs of the 15F7 light chain variable region (SEQ IDNO:17). Optionally, any differences in CDRs of the mature heavy chainvariable region and mature light chain variable region from SEQ IDNOS:16 and 17, respectively reside in positions H60-H65. In somehumanized antibodies at least one of positions L42, L49, and L69 isoccupied by N, S, and K, respectively, and at least one of positions H1,H20, H27, H30, H38, H40, H48, H66, H67, H75, H82A, and H91 is occupiedby E, L, Y, T, K, R, I, K, A, S, R, and F, respectively. In somehumanized antibodies positions L42, L49, and L69 are occupied by N, S,and K, respectively, and H1, H20, H27, H30, H38, H40, H48, H66, H67,H75, H82A, and H91 are occupied by E, L, Y, T, K, R, I, K, A, S, R, andF, respectively. In some humanized antibodies at least one of positionsL8, L45, and L80 is occupied by S, R, and T, respectively. In somehumanized antibodies at least one of positions L8, L45, L80, and L104 isoccupied by S, R, T, and V, respectively. In some humanized antibodiesat least one of positions H41 and H69 is occupied by A and L,respectively. In some humanized antibodies positions L8, L45, and L80are occupied by S, R, and T, respectively. In some humanized antibodiesposition L104 is occupied by V. In some humanized antibodies positionsH41 and H69 are occupied by A and L, respectively. Some humanizedantibodies comprise a mature heavy chain variable region having an aminoacid sequence at least 95% identical to SEQ ID NO:58 and a mature lightchain variable region having an amino acid sequence at least 95%identical to SEQ ID NO:59. In some humanized antibodies, the matureheavy chain variable region has an amino acid sequence of SEQ ID NO:57and the mature light chain variable region has an amino acid sequence ofSEQ ID NO:59. In some humanized antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:57 and themature light chain variable region has an amino acid sequence of SEQ IDNO:60. In some humanized antibodies, the mature heavy chain variableregion has an amino acid sequence of SEQ ID NO:58 and the mature lightchain variable region has an amino acid sequence of SEQ ID NO:59. Insome humanized antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:58 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:60.

Any of the above described antibodies can be an intact antibody, asingle-chain antibody, Fab, or Fab′2 fragment. In any of the antibodies,the mature light chain variable region can be fused to a light chainconstant region and the mature heavy chain variable region can be fusedto a heavy chain constant region. Optionally, the heavy chain constantregion is a mutant form of a natural human heavy chain constant regionwhich has reduced binding to a Fcγ receptor relative to the naturalhuman heavy chain constant region. Optionally the heavy chain constantregion is of IgG1 isotype. Optionally, the mature heavy chain variableregion is fused to a heavy chain constant region having the sequence ofSEQ ID NO:61 and/or the mature light chain variable region is fused to alight chain constant region having the sequence of SEQ ID NO:62.Optionally, the mature heavy chain variable region is fused to a heavychain constant region having the sequence of SEQ ID NO:61, 89, or 101and/or the mature light chain variable region is fused to a light chainconstant region having the sequence of SEQ ID NO:62 or 90. Any of theabove antibodies can be conjugated to a therapeutic or cytotoxic agent,such as saporin or a radioisotope.

The invention further provides pharmaceutical composition comprising anyof the above antibodies and a pharmaceutically acceptable carrier.

The invention further provides a nucleic acid encoding the heavy and/orlight chain(s) of any of the above described antibodies, such as any ofSEQ ID NOS:63-64, 67-68, 71-73, and 77-80. The invention furtherprovides a nucleic acid encoding the heavy and/or light chain(s) of anyof the above described antibodies, such as any of SEQ ID NOS:63-64,67-68, 71-73, 77-80, 92-93, 99-100, or 102.

The invention further provides a recombinant expression vectorcomprising a nucleic acid as above described and a host cell transformedwith such a recombinant expression vector.

The invention further provides a method of humanizing an antibody, themethod comprising: (a) determining the sequences of the heavy and lightchain variable regions of a mouse antibody; (b) synthesizing a nucleicacid encoding a humanized heavy chain comprising CDRs of the mouseantibody heavy chain and a nucleic acid encoding a humanized light chaincomprising CDRs of the mouse antibody light chain; (c) expressing thenucleic acids in a host cell to produce a humanized antibody; whereinthe mouse antibody is 15F7, 6C12, or 13G10.

The invention further provides a method of producing a humanized,chimeric, or veneered antibody, the method comprising: (a) culturingcells transformed with nucleic acids encoding the heavy and light chainsof the antibody, so that the cells secrete the antibody; and (b)purifying the antibody from cell culture media; wherein the antibody isan antibody is a humanized, chimeric, or veneered form of 15F7, 6C12, or13G10.

The invention further provides a method of producing a cell lineproducing a humanized, chimeric, or veneered antibody, the methodcomprising: (a) introducing a vector encoding heavy and light chains ofan antibody and a selectable marker into cells; (b) propagating thecells under conditions to select for cells having increased copy numberof the vector; (c) isolating single cells from the selected cells; and(d) banking cells cloned from a single cell selected based on yield ofantibody; wherein the antibody is a humanized, chimeric, or veneeredform of 15F7, 6C12, or 13G10. Optionally, the method further comprisespropagating the cells under selective conditions and screening for celllines naturally expressing and secreting at least 100 mg/L/10⁶ cells/24h.

The invention further provides a method of treating or effectingprophylaxis of a cancer in a patient having or at risk for the cancer,the method comprising administering to the patient an effective regimeof any of the above described antibodies. In some methods, the patienthas a cancer, and the cancer is melanoma, breast cancer, lung cancer, orcolorectal cancer.

The invention further provides a method of inhibiting cell adhesion in abiological sample, the method comprising contacting the biologicalsample with an effective amount of any of the above describedantibodies. In some methods, the cell adhesion is mediated by the LG4-5modules of the G domain of laminin α4. In some methods, the biologicalsample comprises cancer cells.

The invention further provides a method for detecting the presence of acancer, such as melanoma, in a biological sample, the method comprising:(a) contacting the biological sample with any of the above antibodies;(b) detecting binding of the antibody to the biological sample; (c)contacting a control sample with the antibody; (d) detecting binding ofthe antibody to the control sample; and (e) comparing binding of theantibody to the biological sample with binding of the antibody to thecontrol sample, whereby increased binding of the antibody to thebiological sample compared to the control sample indicates the presenceof cancer in the biological sample. Optionally the control sample andthe biological sample comprise cells of the same tissue origin.Optionally binding of the antibody to the biological sample is at least2-fold or 5-fold greater than the binding of the antibody to the controlsample.

The invention further provides a method of evaluating the efficacy of atherapeutic agent in a patient diagnosed with a cancer, the methodcomprising: (a) contacting a first biological sample from the patient,obtained prior to treatment with the therapeutic agent, with any of theabove antibodies; (b) detecting binding of the antibody to the firstbiological sample; (c) contacting a second biological sample from thepatient, obtained following treatment with the therapeutic agent, withthe antibody; (d) detecting binding of the antibody to the secondbiological sample; (e) comparing binding of the antibody to the firstbiological sample with binding of the antibody to the second biologicalsample, whereby decreased binding of the antibody to the secondbiological sample compared to the first biological sample indicates thatthe therapeutic agent is effective in treating the cancer in thepatient.

The invention further provides a method of inhibiting binding of lamininα4 to a heparan sulfate proteoglycan in a biological sample, the methodcomprising contacting the biological sample with any of the aboveantibodies. Optionally, the heparan sulfate proteoglycan is syndecan-1,-2, -3, or -4 or glypican, a betaglycan, CD44, perlecan, agrin, orcollagen XVIII.

The invention further provides a method of treating or effectingprophylaxis of a disease in which the LG4-5 modules of the G domain oflaminin α4 contribute to progression of the disease, the methodcomprising administering to a patient having or at risk of the diseasean effective regime of any of the above antibodies. Optionally, thedisease is a cancer, such as melanoma, breast cancer, lung cancer, orcolorectal cancer. Optionally the LG4-5 modules of the G domain oflaminin α4 contribute to progression of the disease by means ofinteraction with a heparan sulfate proteoglycan. Optionally, the heparansulfate proteoglycan is syndecan-1, -2, -3, or -4. Optionally, thedisease is psoriasis, sarcoidosis, multiple sclerosis, or psoriatricarthritis.

The invention further provides a method of treating or effectingprophylaxis of an autoimmune disease in a patient having or at risk forthe autoimmune disease, the method comprising administering to thepatient an effective regime of any of the above described antibodies.Optionally, the disease is diabetes, Crohn's disease, ulcerativecolitis, multiple sclerosis, stiff man syndrome, rheumatoid arthritis,myasthenia gravis, systemic lupus erythematosus, celiac disease,psoriasis, psoriatic arthritis, sarcoidosis, ankylosing spondylitis,Sjogren's syndrome, or uveitis.

The invention further provides a method of inhibiting angiogenesis in apatient, the method comprising administering to a patient an effectiveregime of any of the above described antibodies. Optionally the patienthas a cancer.

The invention further provides a method of treating or effectingprophylaxis of obesity or an obesity-related disease in a patient havingor at risk for obesity or the obesity-related disease, the methodcomprising administering to the patient an effective regime of any ofthe above described antibodies. Optionally, the obesity-related diseaseis non-alcoholic steatohepatitis (NASH), Prader-Willi syndrome,craniopharyngioma, Bardet-Biedl syndrome, Cohen syndrome, or MOMOsyndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows binding as assessed by flow cytometry of a LG1-3-specificLAMA4 antibody and the 6C12, 13G10, and 15F7 antibodies to 293 cellsdisplaying LAMA4 fragments containing LG1-5, LG1-3, and LG4-5.

FIG. 2A-C show relative binding and on/off rates for the 15F7, 6C12, and13G10 antibodies, respectively, as assessed by ForteBio.

FIG. 3 shows the capacity of serially diluted 6C12, 13G10 and 15F7antibodies to bind to LAMA4-displaying 293 cells as assessed by flowcytometry.

FIG. 4A-C show assessment of binding by FACS analysis of the 6C12,13G10, and 15F7 antibodies, respectively, to LAMA4-displaying 293 cellsin the presence of decreasing ratios (5:1, 1:1, and 1:5) of competingblocking antibodies.

FIG. 5A-C show staining of tumor microarray slides for human breast,colon, and lung tumors, respectively, with the 15F7 antibody.

FIG. 6 shows a cell adhesion assay in which recombinant LG4-5-coatedELISA plates were incubated with 20 ug/ml 15F7, 6C12, 13G10, and mouseIgG1 control and then assayed for their ability to bind to WM-266-4human melanoma cells.

FIG. 7 shows toxicity to WM-266-4 cells when incubated with the 15F7antibody and a saporin-conjugated anti-mouse secondary antibody.

FIG. 8 shows the capacity of serially diluted chimeric 15F7 andhumanized 15F7 variants H1L1, H1L2, H2L1, and H2L2 to bind toLAMA4-displaying cells as assessed by flow cytometry.

FIG. 9 shows binding as assessed by flow cytometry of chimeric 15F7 andhumanized 15F7 variants H1L1, H1L2, H2L1, and H2L2 to 293 cellsdisplaying LAMA4 fragments containing LG1-5, LG1-3, and LG4-5.

FIG. 10A-B show relative binding and on/off rates for m15F7, chimeric15F7 and humanized 15F7 variants H1L1, H1L2, H2L1, and H2L2 as assessedby ForteBio, with the anti-His sensor being loaded with His-LAMA4followed by association and dissociation of the 15F7 antibodies in 10A,and the goat anti-human Fc sensor being loaded with the antibodiesfollowed by association and dissociation of His-LAMA4 in 10B.

FIG. 11A-B show ratios of the relative levels of pAkt to Akt in humanmelanoma cells treated with laminin 411 or BSA control and with 15F7 ormIgG1 control. FIG. 11A shows the ratio for each individual sample, andFIG. 11B shows averages and standard errors for each group (n=3).

FIGS. 12A-D show exemplary humanized 15F7 Vh designs, with backmutationsand other mutations based on selected human frameworks. The gray-shadedareas in the first column indicate the CDRs as defined by Chothia, andthe gray-shaded areas in the remaining columns indicate the CDRs asdefined by Kabat.

FIGS. 13A-D show exemplary humanized 15F7 Vk designs, with backmutationsand other mutations based on selected human frameworks. The gray-shadedareas in the first column indicate the CDRs as defined by Chothia, andthe gray-shaded areas in the remaining columns indicate the CDRs asdefined by Kabat.

BRIEF DESCRIPTION OF THE SEQUENCES

The nucleotide and amino acid sequences listed in the accompanyingsequence listing are shown using standard letter abbreviations fornucleotide bases, and three-letter code for amino acids. The nucleotidesequences follow the standard convention of beginning at the 5′ end ofthe sequence and proceeding forward (i.e., from left to right in eachline) to the 3′ end. Only one strand of each nucleotide sequence isshown, but the complementary strand is understood to be included by anyreference to the displayed strand. The amino acid sequences follow thestandard convention of beginning at the amino terminus of the sequenceand proceeding forward (i.e., from left to right in each line) to thecarboxy terminus.

SEQ ID NO:1 sets forth the amino acid sequence of laminin α4 as providedby UniProt Number Q16363.

SEQ ID NO:2 sets forth the amino acid sequence of laminin α4 as providedby GenBank Accession Number NP001098676.

SEQ ID NO:3 sets forth the amino acid sequence of laminin α4 as providedby GenBank Accession Number NP001098677.

SEQ ID NO:4 sets forth the amino acid sequence of the G domain oflaminin α4.

SEQ ID NO:5 sets forth the amino acid sequence of the LG1 module of theG domain of laminin α4.

SEQ ID NO:6 sets forth the amino acid sequence of the LG2 module of theG domain of laminin α4.

SEQ ID NO:7 sets forth the amino acid sequence of the LG3 module of theG domain of laminin α4.

SEQ ID NO:8 sets forth the amino acid sequence of the LG1-3 modules ofthe G domain of laminin α4.

SEQ ID NO:9 sets forth the amino acid sequence of the LG4 module of theG domain of laminin α4.

SEQ ID NO:10 sets forth the amino acid sequence of the LG5 module of theG domain of laminin α4.

SEQ ID NO:11 sets forth the amino acid sequence of the LG4-5 modules ofthe G domain of laminin α4.

SEQ ID NO:12 sets forth the amino acid sequence of syndecan-1 asprovided by UniProt Number P18827.

SEQ ID NO:13 sets forth the amino acid sequence of syndecan-2 asprovided by UniProt Number P34741.

SEQ ID NO:14 sets forth the amino acid sequence of syndecan-3 asprovided by UniProt Number 075056.

SEQ ID NO:15 sets forth the amino acid sequence of syndecan-4 asprovided by UniProt Number P31431.

SEQ ID NO:16 sets forth the amino acid sequence of mouse 15F7 matureheavy chain variable region.

SEQ ID NO:17 sets forth the amino acid sequence of mouse 15F7 maturelight chain variable region.

SEQ ID NO:18 sets forth the amino acid sequence of the 15F7 heavy chainvariable region signal peptide.

SEQ ID NO:19 sets forth the amino acid sequence of the 15F7 light chainvariable region signal peptide.

SEQ ID NO:20 sets forth the amino acid sequence of CDR1, as defined byKabat, of the mouse 15F7 heavy chain.

SEQ ID NO:21 sets forth the amino acid sequence of CDR2, as defined byKabat, of the mouse 15F7 heavy chain.

SEQ ID NO:22 sets forth the amino acid sequence of CDR3, as defined byKabat, of the mouse 15F7 heavy chain.

SEQ ID NO:23 sets forth the amino acid sequence of CDR1, as defined byKabat, of the mouse 15F7 light chain.

SEQ ID NO:24 sets forth the amino acid sequence of CDR2, as defined byKabat, of the mouse 15F7 light chain.

SEQ ID NO:25 sets forth the amino acid sequence of CDR3, as defined byKabat, of the mouse 15F7 light chain.

SEQ ID NO:26 sets forth the amino acid sequence of mouse 6C12 matureheavy chain variable region.

SEQ ID NO:27 sets forth the amino acid sequence of mouse 6C12 maturelight chain variable region, version 1.

SEQ ID NO:28 sets forth the amino acid sequence of the 6C12 heavy chainvariable region signal peptide.

SEQ ID NO:29 sets forth the amino acid sequence of the 6C12 light chainvariable region signal peptide.

SEQ ID NO:30 sets forth the amino acid sequence of CDR1, as defined byKabat, of the mouse 6C12 heavy chain.

SEQ ID NO:31 sets forth the amino acid sequence of CDR2, as defined byKabat, of the mouse 6C12 heavy chain.

SEQ ID NO:32 sets forth the amino acid sequence of CDR3, as defined byKabat, of the mouse 6C12 heavy chain.

SEQ ID NO:33 sets forth the amino acid sequence of CDR1, as defined byKabat, of the mouse 6C12 light chain, version 1.

SEQ ID NO:34 sets forth the amino acid sequence of CDR2, as defined byKabat, of the mouse 6C12 light chain.

SEQ ID NO:35 sets forth the amino acid sequence of CDR3, as defined byKabat, of the mouse 6C12 light chain.

SEQ ID NO:36 sets forth the amino acid sequence of mouse 13G10 matureheavy chain variable region, version 1.

SEQ ID NO:37 sets forth the amino acid sequence of mouse 13G10 matureheavy chain variable region, version 2.

SEQ ID NO:38 sets forth the amino acid sequence of mouse 13G10 maturelight chain variable region.

SEQ ID NO:39 sets forth the amino acid sequence of the 13G10 heavy chainvariable region signal peptide, version 1.

SEQ ID NO:40 sets forth the amino acid sequence of the 13G10 heavy chainvariable region signal peptide, version 2.

SEQ ID NO:41 sets forth the amino acid sequence of the 13G10 light chainvariable region signal peptide.

SEQ ID NO:42 sets forth the amino acid sequence of CDR1, as defined byKabat, of the mouse 13G10 heavy chain, version 1.

SEQ ID NO:43 sets forth the amino acid sequence of CDR2, as defined byKabat, of the mouse 13G10 heavy chain, version 1.

SEQ ID NO:44 sets forth the amino acid sequence of CDR3, as defined byKabat, of the mouse 13G10 heavy chain, version 1.

SEQ ID NO:45 sets forth the amino acid sequence of CDR1, as defined byKabat, of the mouse 13G10 heavy chain, version 2.

SEQ ID NO:46 sets forth the amino acid sequence of CDR2, as defined byKabat, of the mouse 13G10 heavy chain, version 2.

SEQ ID NO:47 sets forth the amino acid sequence of CDR3, as defined byKabat, of the mouse 13G10 heavy chain, version 2.

SEQ ID NO:48 sets forth the amino acid sequence of CDR1, as defined byKabat, of the mouse 13G10 light chain.

SEQ ID NO:49 sets forth the amino acid sequence of CDR2, as defined byKabat, of the mouse 13G10 light chain.

SEQ ID NO:50 sets forth the amino acid sequence of CDR3, as defined byKabat, of the mouse 13G10 light chain.

SEQ ID NO:51 sets forth the amino acid sequence of a human VH acceptorFR as provided by NCBI Accession Code ACF36857.1, version 1.

SEQ ID NO:52 sets forth the amino acid sequence of a human VH acceptorFR as provided by NCBI Accession Code BAC01530.1, version 1.

SEQ ID NO:53 sets forth the amino acid sequence of a human VL acceptorFR as provided by NCBI Accession Code AAY33350.1.

SEQ ID NO:54 sets forth the amino acid sequence of a human VL acceptorFR as provided by NCBI Accession Code BAC01583.1, version 1.

SEQ ID NO:55 sets forth the amino acid sequence of humanized 15F7 heavychain variable region with no backmutations or other mutations.

SEQ ID NO:56 sets forth the amino acid sequence of humanized 15F7 lightchain variable region with no backmutations or other mutations.

SEQ ID NO:57 sets forth the amino acid sequence of humanized 15F7 heavychain variable region version 1 (H1).

SEQ ID NO:58 sets forth the amino acid sequence of humanized 15F7 heavychain variable region version 2 (H2).

SEQ ID NO:59 sets forth the amino acid sequence of humanized 15F7 lightchain variable region version 1 (L1).

SEQ ID NO:60 sets forth the amino acid sequence of humanized 15F7 lightchain variable region version 2 (L2).

SEQ ID NO:61 sets forth the amino acid sequence of an exemplary humanIgG1 constant region.

SEQ ID NO:62 sets forth the amino acid sequence of an exemplary humankappa light chain constant region without a N-terminal arginine.

SEQ ID NO:63 sets forth the nucleic acid sequence of the murine 15F7heavy chain variable region.

SEQ ID NO:64 sets forth the nucleic acid sequence of the murine 15F7light chain variable region version.

SEQ ID NO:65 sets forth the nucleic acid sequence of the 15F7 heavychain variable region signal peptide.

SEQ ID NO:66 sets forth the nucleic acid sequence of the 15F7 lightchain variable region signal peptide.

SEQ ID NO:67 sets forth the nucleic acid sequence of the murine 6C12heavy chain variable region.

SEQ ID NO:68 sets forth the nucleic acid sequence of the murine 6C12light chain variable region, version 1.

SEQ ID NO:69 sets forth the nucleic acid sequence of the 6C12 heavychain variable region signal peptide.

SEQ ID NO:70 sets forth the nucleic acid sequence of the 6C12 lightchain variable region signal peptide.

SEQ ID NO:71 sets forth the nucleic acid sequence of the murine 13G10heavy chain variable region, version 1.

SEQ ID NO:72 sets forth the nucleic acid sequence of the murine 13G10heavy chain variable region, version 2.

SEQ ID NO:73 sets forth the nucleic acid sequence of the murine 13G10light chain variable region.

SEQ ID NO:74 sets forth the nucleic acid sequence of the 13G10 heavychain variable region signal peptide, version 1.

SEQ ID NO:75 sets forth the nucleic acid sequence of the 13G10 heavychain variable region signal peptide, version 2.

SEQ ID NO:76 sets forth the nucleic acid sequence of the 13G10 lightchain variable region signal peptide.

SEQ ID NO:77 sets forth the nucleic acid sequence of humanized 15F7heavy chain variable region version 1 (H1).

SEQ ID NO:78 sets forth the nucleic acid sequence of humanized 15F7heavy chain variable region version 2 (H2).

SEQ ID NO:79 sets forth the nucleic acid sequence of humanized 15F7light chain variable region version 1 (L1), version 1.

SEQ ID NO:80 sets forth the nucleic acid sequence of humanized 15F7light chain variable region version 2 (L2).

SEQ ID NO:81 sets forth the nucleic acid sequence of the G domain oflaminin α4.

SEQ ID NO:82 sets forth the nucleic acid sequence of the LG1 module ofthe G domain of laminin α4.

SEQ ID NO:83 sets forth the nucleic acid sequence of the LG2 module ofthe G domain of laminin α4.

SEQ ID NO:84 sets forth the nucleic acid sequence of the LG3 module ofthe G domain of laminin α4.

SEQ ID NO:85 sets forth the nucleic acid sequence of the LG1-3 modulesof the G domain of laminin α4.

SEQ ID NO:86 sets forth the nucleic acid sequence of the LG4 module ofthe G domain of laminin α4.

SEQ ID NO:87 sets forth the nucleic acid sequence of the LG5 module ofthe G domain of laminin α4.

SEQ ID NO:88 sets forth the nucleic acid sequence of the LG4-5 modulesof the G domain of laminin α4.

SEQ ID NO:89 sets forth the amino acid sequence of an exemplary humanIgG1 constant region of the IgG1 G1m3 allotype.

SEQ ID NO:90 sets forth the amino acid sequence of an exemplary humankappa light chain constant region with a N-terminal arginine.

SEQ ID NO:91 sets forth the amino acid sequence of an exemplary humanIgG1 constant region without a C-terminal lysine.

SEQ ID NO:92 sets forth the nucleic acid sequence of an exemplary humanIgG1 constant region of the IgG1 G1m3 allotype.

SEQ ID NO:93 sets forth the nucleic acid sequence of an exemplary humankappa light chain constant region with a N-terminal arginine.

SEQ ID NO:94 sets forth the amino acid sequence of mouse 6C12 maturelight chain variable region, version 2.

SEQ ID NO:95 sets forth the amino acid sequence of CDR1, as defined byKabat, of the mouse 6C12 light chain, version 2.

SEQ ID NO:96 sets forth the amino acid sequence of a human VH acceptorFR as provided by NCBI Accession Code ACF36857.1, version 2.

SEQ ID NO:97 sets forth the amino acid sequence of a human VH acceptorFR as provided by NCBI Accession Code BAC01530.1, version 2.

SEQ ID NO:98 sets forth the amino acid sequence of a human VL acceptorFR as provided by NCBI Accession Code BAC01583.1, version 2.

SEQ ID NO:99 sets forth the nucleic acid sequence of the murine 6C12light chain variable region, version 2.

SEQ ID NO:100 sets forth the nucleic acid sequence of humanized 15F7light chain variable region version 1 (L1), version 2.

SEQ ID NO:101 sets forth the amino acid sequence of an exemplary humanIgG1 constant region of the IgG1 Glm3 allotype.

SEQ ID NO:102 sets forth the nucleic acid sequence of an exemplary humankappa light chain constant region without a N-terminal arginine.

Definitions

Monoclonal antibodies or other biological entities are typicallyprovided in isolated form. This means that an antibody or otherbiologically entity is typically at least 50% w/w pure of interferingproteins and other contaminants arising from its production orpurification but does not exclude the possibility that the monoclonalantibody is combined with an excess of pharmaceutically acceptablecarrier(s) or other vehicle intended to facilitate its use. Sometimesmonoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or 99% w/wpure of interfering proteins and contaminants from production orpurification. Often an isolated monoclonal antibody or other biologicalentity is the predominant macromolecular species remaining after itspurification.

Specific binding of an antibody to its target antigen means an affinityof at least 10⁶, 10⁷, 10 ⁸, 10⁹, or 10¹⁰ M⁻¹. Specific binding isdetectably higher in magnitude and distinguishable from non-specificbinding occurring to at least one unrelated target. Specific binding canbe the result of formation of bonds between particular functional groupsor particular spatial fit (e.g., lock and key type) whereas nonspecificbinding is usually the result of van der Waals forces. Specific bindingdoes not however necessarily imply that an antibody binds one and onlyone target.

The basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. This variable region is initially expressed linkedto a cleavable signal peptide. The variable region without the signalpeptide is sometimes referred to as a mature variable region. Thus, forexample, a light chain mature variable region means a light chainvariable region without the light chain signal peptide. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 or more amino acids. See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7 (incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form theantibody binding site. Thus, an intact antibody has two binding sites.Except in bifunctional or bispecific antibodies, the two binding sitesare the same. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of Kabat,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabatalso provides a widely used numbering convention (Kabat numbering) inwhich corresponding residues between different heavy chains or betweendifferent light chains are assigned the same number.

The term “antibody” includes intact antibodies and binding fragmentsthereof. Typically, fragments compete with the intact antibody fromwhich they were derived for specific binding to the target includingseparate heavy chains, light chains Fab, Fab′, F(ab′)₂, F(ab)c, Dabs,nanobodies, and Fv. Fragments can be produced by recombinant DNAtechniques, or by enzymatic or chemical separation of intactimmunoglobulins. The term “antibody” also includes a bispecific antibodyand/or a humanized antibody. A bispecific or bifunctional antibody is anartificial hybrid antibody having two different heavy/light chain pairsand two different binding sites (see, e.g., Songsivilai and Lachmann,Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol.,148:1547-53 (1992)). In some bispecific antibodies, the two differentheavy/light chain pairs include a humanized 15F7 heavy chain/light chainpair and a heavy chain/light chain pair specific for a different epitopeon laminin α4 than that bound by 15F7.

In some bispecific antibodies, one heavy chain light chain pair is ahumanized 15F7 antibody as further disclosed below and the heavy lightchain pair is from an antibody that binds to a receptor expressed on theblood brain barrier, such as an insulin receptor, an insulin-like growthfactor (IGF) receptor, a leptin receptor, or a lipoprotein receptor, ora transferrin receptor (Friden et al., PNAS 88:4771-4775, 1991; Fridenet al., Science 259:373-377, 1993). Such a bispecific antibody can betransferred cross the blood brain barrier by receptor-mediatedtranscytosis. Brain uptake of the bispecific antibody can be furtherenhanced by engineering the bi-specific antibody to reduce its affinityto the blood brain barrier receptor. Reduced affinity for the receptorresulted in a broader distributioin in the brain (see, e.g., Atwal. etal., Sci. Trans. Med. 3, 84ra43, 2011; Yu et al., Sci. Trans. Med. 3,84ra44, 2011).

Exemplary bispecific antibodies can also be (1) a dual-variable-domainantibody (DVD-Ig), where each light chain and heavy chain contains twovariable domains in tandem through a short peptide linkage (Wu et al.,Generation and Characterization of a Dual Variable Domain Immunoglobulin(DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg(2010)); (2) a Tandab, which is a fusion of two single chain diabodiesresulting in a tetravalent bispecific antibody that has two bindingsites for each of the target antigens; (3) a flexibody, which is acombination of scFvs with a diabody resulting in a multivalent molecule;(4) a so called “dock and lock” molecule, based on the “dimerization anddocking domain” in Protein Kinase A, which, when applied to Fabs, canyield a trivalent bispecific binding protein consisting of two identicalFab fragments linked to a different Fab fragment; (5) a so-calledScorpion molecule, comprising, e.g., two scFvs fused to both termini ofa human Fc-region. Examples of platforms useful for preparing bispecificantibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2(F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab armexchange, Genmab).

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids (also known aslinear epitopes) are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding (also known asconformational epitopes) are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined X-ray crystallography of theantibody bound to its antigen to identify contact residues.Alternatively, two antibodies have the same epitope if all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies haveoverlapping epitopes if some amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50% as measured in acompetitive binding assay. Some test antibodies inhibit binding of thereferences antibody by at least 75%, 90% or 99%. Antibodies identifiedby competition assay (competing antibodies) include antibodies bindingto the same epitope as the reference antibody and antibodies binding toan adjacent epitope sufficiently proximal to the epitope bound by thereference antibody for steric hindrance to occur.

The term “pharmaceutically acceptable” means that the carrier, diluent,excipient, or auxiliary is compatible with the other ingredients of theformulation and not substantially deleterious to the recipient thereof.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

An individual is at increased risk of a disease if the subject has atleast one known risk-factor (e.g., genetic, biochemical, family history,and situational exposure) placing individuals with that risk factor at astatistically significant greater risk of developing the disease thanindividuals without the risk factor.

The term “biological sample” refers to a sample of biological materialwithin or obtainable from a biological source, for example a human ormammalian subject. Such samples can be organs, organelles, tissues,sections of tissues, bodily fluids, peripheral blood, blood plasma,blood serum, cells, molecules such as proteins and peptides, and anyparts or combinations derived therefrom. The term biological sample canalso encompass any material derived by processing the sample. Derivedmaterial can include cells or their progeny. Processing of thebiological sample may involve one or more of filtration, distillation,extraction, concentration, fixation, inactivation of interferingcomponents, and the like.

The term “control sample” refers to a biological sample not known orsuspected to include cancerous cells, or at least not known or suspectto include cancerous cells of a given type. Control samples can beobtained from individuals not afflicted with cancer or a specificallychosen type of cancer. Alternatively, control samples can be obtainedfrom patients afflicted with cancer or a specifically chosen type ofcancer. Such samples can be obtained at the same time as a biologicalsample thought to comprise the cancer or on a different occasion. Abiological sample and a control sample can both be obtained from thesame tissue (e.g., a tissue section containing both tumor tissue andsurrounding normal tissue). Preferably, control samples consistessentially or entirely of normal, healthy cells and can be used incomparison to a biological sample thought to comprise cancer cells or aparticular type of cancer cells. Preferably, the cells in the controlsample have the same tissue origin as the cancer cells thought to be inthe biological sample (e.g., lung or brain). Preferably, the cancercells thought to be in the biological sample arise from the same celltype (e.g., neuronal, epithelial, mesenchymal, hematopoietic) as thetype of cells in the control sample.

The term “disease” refers to any abnormal condition that impairsphysiological function. The term is used broadly to encompass anydisorder, illness, abnormality, pathology, sickness, condition, orsyndrome in which physiological function is impaired, irrespective ofthe nature of the etiology.

The term “symptom” refers to a subjective evidence of a disease, such asaltered gait, as perceived by the subject. A “sign” refers to objectiveevidence of a disease as observed by a physician.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): trp, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Nonconservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention. After alignment, ifa subject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

Compositions or methods “comprising” or “including” one or more recitedelements may include other elements not specifically recited. Forexample, a composition that “comprises” or “includes” an antibody maycontain the antibody alone or in combination with other ingredients.

Designation of a range of values includes all integers within ordefining the range, and all subranges defined by integers within therange.

Unless otherwise apparent from the context, the term “about” encompassesvalues within a standard margin of error of measurement (e.g., SEM) of astated value.

Statistical significance means p≤0.05.

The singular forms of the articles “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” can include a pluralityof compounds, including mixtures thereof.

DETAILED DESCRIPTION I. General

The invention provides antibodies that specifically bind to the LG4-5modules of the G domain of laminin α4. The antibodies have the abilityselectively to stain cancer cells relative to normal tissue. Theantibodies also have the ability to inhibit laminin-α4-mediated canceror tumor cell adhesion. The antibodies can be used for detecting cancer,evaluating the efficacy of a cancer therapy, treating cancer, andtreating obesity or obesity-related diseases, among other applications.

II. Target Molecules

Laminins are a family of extracellular matrix glycoproteins and are themajor non-collagenous constitutent of basement membranes. They have beenreported to be involved in biological processes including cell adhesion,differentiation, migration, signaling, neurite outgrowth, andmetastasis, among other processes. Laminins are heterotrimeric proteinsof three chains: an alpha chain, a beta chain, and a gamma chain. Thethree chains form a cruciform structure consisting of three short arms,each formed by a different chain, and a long arm composed of all threechains. In mammals, five different alpha chains, three different betachains, and three different gamma chains have been identified that canassemble into fifteen different heterotrimeric combinations.

The laminin alpha chains have a large C-terminal globular domain (Gdomain) that has five tandem homologous laminin G-like modules (LG1-5)of about 200 amino acids. For example, the G domain of laminin α4 isdefined by UniProt sequence Q16363 as amino acid positions 833-1820 (SEQID NO:4), and the five LG modules of laminin α4 are defined by UniProtsequence Q16363 as follows: LG1 (SEQ ID NO:5) includes amino acidpositions 833-1035, LG2 (SEQ ID NO:6) includes amino acid positions1047-1227, LG3 (SEQ ID NO:7) includes amino acid positions 1234-1402,LG4 (SEQ ID NO:9) includes amino acid positions 1469-1640, and LG5 (SEQID NO:10) includes amino acid positions 1647-1820. In some cases, the Gdomain can be SEQ ID NO:4; in other cases it can include amino acidpositions 833-1820 of UniProt sequence Q16363. In some cases, the LG1module can be SEQ ID NO:5; in other cases it can include amino acidpositions 833-1035 of UniProt sequence Q16363. In some cases, the LG2module can be SEQ ID NO:6; in other cases it can include amino acidpositions 1047-1227 of UniProt sequence Q16363. In some cases, the LG3module can be SEQ ID NO:7; in other cases it can include amino acidpositions 1234-1402 of UniProt sequence Q16363. In some cases, the LG4module can be SEQ ID NO:9; in other cases it can include amino acidpositions 1469-1640 of UniProt sequence Q16363. In some cases, the LG5module can be SEQ ID NO:10; in other cases it can include amino acidpositions 1647-1820 of UniProt sequence Q16363. The LG1-3 modules (SEQID NO:8) are connected to the LG4-5 modules (SEQ ID NO:11) by a linkerdomain. The laminin α4 chain (also known as LAMA4, laminin subunit α4,laminin-14 subunit alpha, laminin-8 subunit alpha, and laminin-9 subunitalpha) is 200 kDa and is the shortest variant. Compared to the α1, α2,and α5 chains, laminin α4 has a truncated N-terminus. Laminin α4 iswidely distributed both in adults and during development. It is presentin laminin-8 (laminin 411 or alpha4/beta1/gamma1), laminin-9 (laminin421 or alpha4/beta2/gamma1), and laminin-14 (laminin 411 oralpha4/beta1/gamma1).

Unless otherwise apparent from context, reference to laminin α4 or itsfragments, domains, or modules includes the natural human amino acidsequences including isoforms and allelic variants thereof. Exemplaryhuman sequences are designated UniProt Number Q16363 and GenBankAccession Numbers NP001098676 and NP001098677 (SEQ ID NOS:1, 2, and 3,respectively). Some antibodies bind to an epitope within the LG4-5modules of the G domain of laminin α4. The epitope can be in LG4, inLG5, or split so that residues forming the epitope come from both LG4and LG5.

Laminin α4 has been reported to be a binding partner for syndecans. Forexample, cells overexpressing syndecan-2 or -4 have been reported tobind to the LG4 module of the G domain of laminin α4. See Matsuura etal., J. Invest. Dermatol. 122:614-620 (2004). Syndecans aretransmembrane heparan sulfate proteoglycan receptors that constitute themajor physiological form of heparan sulfate on the cell surface. Thereare four reported members of the syndecan protein family: syndecan-1(also known as SYND1, SDC1, SDC, or CD138), syndecan-2 (also known asSYND2, SDC2, fibroglycan, heparan sulfate proteoglycan core protein,HSPG, HSPG1, and CD362), syndecan-3 (also known as SYND3 and SDC3), andsyndecan-4 (also known as SYND4, SDC4, amphiglycan, and ryudocan coreprotein). Syndecans have a short cytoplasmic domain, a single-spantransmembrane domain, and an extracellular domain. They have beenreported to bind to growth factors, cytokines, and extracellular matrixproteins. Syndecans have many reported biological roles includingregulating cell growth, differentiation, angiogenesis, and adhesion,among other processes. Unless otherwise apparent from context, referenceto a syndecan or its fragments or domains includes the natural humanamino acid sequences including isoforms and allelic variants thereof.Exemplary human sequences are designated UniProt Numbers P18827(syndecan-1), P34741 (syndecan-2), O75056 (syndecan-3), and P31431(syndecan-4) (SEQ ID NOS:12-15, respectively).

The LG4 and LG5 modules of the G domain of laminin α4 also bind to otherheparan sulfate proteoglycans including cell surface proteins such asglypicans, betaglycans, and CD44 (e.g., Swiss Prot. P16070), and matrixproteins such as perlecan (e.g., Swiss Prot. P98160), agrin (e.g., SwissProt. Q00468), and collagen XVIII (e.g., Swiss Prot. P39060). Binding ofthe LG4-5 modules of the G domain of laminin α4 to any or all of theseor other proteoglycans may contribute to its growth promotingcharacteristics, inflammation, angiogenesis, and growth and metastasisof cancers.

III. Cancers

The above target molecules are involved in the development and/orprogression of cancers. Cancer is a physiological condition in mammalsthat is typically characterized by unregulated cell growth andproliferation. Cancers can be hematopoietic malignancies or solidtumors, i.e., masses of cells that result from excessive cell growth orproliferation, including pre-cancerous legions. Metastatic cancer refersto a cancer that has spread from the place where it first started toanother place in the body. Tumors formed by metastatic cancer cells arecalled a metastatic tumor or a metastasis, which is a term also used torefer to the process by which cancer cells spread to other parts of thebody. In general, metastatic cancer has the same name and same type ofcancer cells as the original, or primary, cancer. Examples solid tumorsinclude melanoma, carcinoma, blastoma, and sarcoma. Hematologicmalignancies include leukemia or lymphoid malignancies, such aslymphoma. More particular examples of such cancers include squamous cellcancer or carcinoma, lung cancer, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioma, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial cancer or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

IV. Antibodies

A. Binding Specificity and Functional Properties

The invention provides antibodies binding to epitopes within the LG4-5modules of the G domain of laminin α4. For example, as defined bylaminin α4 UniProt sequence Q16363, LG4 (SEQ ID NO:9) includes aminoacid positions 1469-1640, LG5 (SEQ ID NO:10) includes amino acidpositions 1647-1820, and LG4-5 (SEQ ID NO:11) includes amino acidpositions 1469-1820. The epitope can be in LG4, in LG5, or split so thatresidues forming the epitope come from both LG4 and LG5. The epitope canbe in particular segments within LG4-5, such as segments from laminin α4UniProt sequence Q16363 ranging from positions 1469-1519, 1520-1570,1571-1621, 1622-1672, 1673-1723, 1724-1774, and 1775-1820. The epitopecan be linear, such as an epitope of, for example, 2-5, 3-5, 3-10, 3-15,3-20, 5-10, 5-15, or 5-20 contiguous amino acids from LG4, LG5, LG4-5,or any of the segments or pairs of adjoining segments specified above.The epitope can also be a conformational epitope including, for example,2-5, 3-5, 3-10, 3-15, 3-20, 5-10, 5-15, or 5-20 non-contiguous aminoacids from any combination of LG4, LG5, LG4-5, and any of the segmentsspecified above.

Antibodies designated 15F7, 6C12, and 13G10 are three such exemplarymouse antibodies. These three monoclonal antibodies each specificallybind to an epitope within the LG4 and/or LG5 modules of the G domain oflaminin α4. These antibodies are further characterized by their lack ofsignificant binding to the LG1-3 modules of the G domain of laminin α4(e.g., same within experimental error as an irrelevant control antibody,or binding that is at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-foldless (e.g., as measured by a flow cytometric binding assay) than anantibody specific for the LG1-3 modules). Some antibodies arecharacterized by their lack of significant binding to other lamininalpha chains, e.g., laminin α1, laminin α2, laminin α3, and laminin α5(e.g., same within experimental error as an irrelevant control antibody,or binding that is at least 2-fold, 3-fold, 4-fold, 5-fold, or 10-foldless (e.g., as measured by a flow cytometric binding assay) than anantibody specific for the relevant other laminin alpha chain). Someantibodies are further characterized by their capacity selectively tobind a biological sample comprising cancer cells (e.g., a sample oftumor tissue) when compared to control sample (e.g., binding of theantibody to the biological sample is at least 1.5-fold, 2-fold, 5-fold,or 10-fold greater than binding of the antibody to the control sample),as shown in Example 2 with melanoma samples. Preferably, the controlsample and the biological sample comprise cells of the same tissueorigin. Ability to bind to specific proteins, modules, or domains can bedemonstrated using exemplary assay formats provided in the examples.Likewise, ability to selectively bind and stain tumor tissue can bedemonstrated using exemplary assay formats provided in the examples.

Some antibodies binding within LG4-5 lack capacity to inhibit binding oflaminin α4 to MCAM (i.e., extent of inhibition the same withinexperimental error as an irrelevant control antibody). Some antibodiesinhibit binding of laminin α4 to a syndecan, such as syndecan-1,syndecan-2, syndecan-3, or syndecan-4. Some antibodies inhibit bindingof laminin α4 to other heparan sulfate proteoglycans including severalother cell surface proteins including any or all of glypicans,betaglycans, and CD44, and matrix proteins such as perlecan, agrin, andcollagen XVIII. Some of the disclosed antibodies can inhibit binding ofthe LG4-5 modules of the G domain of laminin α4 to any or all of theseproteoglycans, and consequently or otherwise inhibit angiogenesis,inflammation and growth and metastasis of cancers.

Inhibition of binding of laminin α4 to a binding partner, for example asyndecan or other proteoglycan, can be tested in a binding assay inwhich an antibody is pre-incubated with recombinant laminin α4 protein,laminin-α4-positive tissue, or laminin-α4-displaying cells, after whichrecombinant proteoglycan or proteoglycan-expressing cells are thenassessed for their ability to bind to laminin α4. Optionally, inhibitionof a test antibody can be demonstrated in comparison to an irrelevantcontrol antibody not binding within the LG4-5 modules of the G domain oflaminin α4 or in comparison to vehicle lacking any antibody.

Some antibodies have the capacity to inhibit laminin-α4-mediated celladhesion of cancer cells, preferably cell adhesion mediated by the LG4-5modules of the G domain of laminin α4. An exemplary cell adhesion assayis described in the examples.

Some antibodies also have the capacity to inhibit laminin-α4-inducedpAkt activation. An exemplary assay is described in the examples.

Inhibition means an inhibition of at least 10%, 20%, 25%, 30%, 40%, 50%,or 75%, (e.g., 10%-75% or 30%-70%) of binding, cell adhesion and/orother functional activity mediated by laminin α4, either alone or incombination with a syndecan or anything else contributing (e.g., otherproteoglycan) to any of its functional activities. Inhibition canusually demonstrated when the antibody is present at a concentration ofabout 20 ug/ml. The antibodies can show inhibition of at least 30% oflaminin-α4-mediated cell adhesion, such as, for example, cell adhesionmediated by the LG4-5 modules of the G domain of laminin α4.

Preferred antibodies inhibit cancer development or progression, or anyaspect thereof, such as metastasis, as shown in an animal model orclinical trial. Animal models of cancer in which human cancer cells areinjected into an immunodeficient laboratory animal, such as a mouse orrat, or transgenic models in which a laboratory animal expresses a humanoncogene or has a knocked out tumor suppressor gene, are widelyavailable. In addition, cell-based assays for particular characteristicsof cancer cells, such as proliferation assays, growth assays, survivalassays, migration assays, invasion assays, and others, are widelyavailable.

Some antibodies bind to the same or overlapping epitope as an antibodydesignated 15F7, 6C12, or 13G10. The sequences of the heavy and lightchain mature variable regions of these antibodies are designated SEQ IDNOS:16 and 17, 26 and 27, and 36/37 and 38, respectively. Anotherversion of the light chain mature variable region of 6C12 is SEQ IDNO:94. Other antibodies having such a binding specificity can beproduced by immunizing mice with laminin α4, or a portion thereofincluding the desired epitope, and screening resulting antibodies forbinding to the LG4-5 modules of the G domain of laminin α4, optionallyin competition with 15F7, 6C12, or 13G10. Antibodies identified by suchassays can then be screened for ability to specifically bind to theLG4-5 modules but not the LG1-3 modules of the G domain of laminin α4 asdescribed in the examples or otherwise. Antibodies can also be screenedfor ability to selectively stain tumor tissue as described in theexamples or otherwise. Antibodies can also be screened for ability toinhibit laminin-α4-mediated tumor cell adhesion as described in theexamples or otherwise.

Antibodies binding to an epitope that includes one or more specifiedresidues can be generated by immunizing with a fragment of laminin α4that includes these one or more residues. The fragment can, for example,have no more than 100, 50, 25, 10 or 5 contiguous amino acids from SEQID NO:11. Such fragments usually have at least 5, 6, 7, 8 or 9contiguous residues of SEQ ID NO:11. The fragments can be linked to acarrier that helps elicit an antibody response to the fragment and/or becombined with an adjuvant that helps elicit such a response.Alternatively, antibodies binding to a desired residue can be obtainedby immunizing with a full-length laminin α4 (SEQ ID NO:1) or thefull-length G domain of laminin α4 (SEQ ID NO:4) or the LG4-5 modules ofthe G domain of laminin α4 (SEQ ID NO:11) or fragments of any of these.Such antibodies can then be screened for differential binding toversions of laminin α4 containing different LG modules of the G domain,such as LG1-3, LG1-5, LG4-5, LG4, or LG5 (SEQ ID NOS:8, 4, 11, 9, and10, respectively) or differential binding to wild type laminin α4compared with mutants of specified residues. The screen against versionsof laminin α4 with different LG modules of the G domain maps antibodybinding to certain LG modules within the G domain of laminin α4. Thescreen against mutants more precisely defines the binding specificity toallow identification of antibodies whose binding is inhibited bymutagenesis of particular residues and which are likely to shareinhibitor properties of other exemplified antibodies.

Human antibodies having the binding specificity of a selected murineantibody (e.g., 15F7, 6C12, or 13G10) can also be produced using avariant of the phage display method. See Winter, WO 92/20791. Thismethod is particularly suitable for producing human antibodies. In thismethod, either the heavy or light chain variable region of the selectedmurine antibody is used as a starting material. If, for example, a lightchain variable region is selected as the starting material, a phagelibrary is constructed in which members display the same light chainvariable region (i.e., the murine starting material) and a differentheavy chain variable region. The heavy chain variable regions can forexample be obtained from a library of rearranged human heavy chainvariable regions. A phage showing strong specific binding for the LG4-5modules of the G domain of laminin α4 (e.g., at least 10⁸ and preferablyat least 10⁹ M⁻¹) is selected. The heavy chain variable region from thisphage then serves as a starting material for constructing a furtherphage library. In this library, each phage displays the same heavy chainvariable region (i.e., the region identified from the first displaylibrary) and a different light chain variable region. The light chainvariable regions can be obtained for example from a library ofrearranged human variable light chain regions. Again, phage showingstrong specific binding for the LG4-5 modules of the G domain of lamininα4 are selected. The resulting antibodies usually have the same orsimilar epitope specificity as the murine starting material.

Other antibodies can be obtained by mutagenesis of cDNA encoding theheavy and light chains of an exemplary antibody, such as 15F7, 6C12, or13G10. Monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%,97%, 98%, or 99% identical to 15F7, 6C12, or 13G10 in amino acidsequence of the mature heavy and/or light chain variable regions andmaintain its functional properties, and/or which differ from therespective antibody by a small number of functionally inconsequentialamino acid substitutions (e.g., conservative substitutions), deletions,or insertions are also included in the invention. Monoclonal antibodieshaving at least one or all six CDR(s) as defined by Kabat that are 90%,95%, 99% or 100% identical to corresponding CDRs of 15F7, 6C12, or 13G10are also included.

The invention also provides antibodies having some or all (e.g., 3, 4,5, and 6) CDRs entirely or substantially from 15F7, 6C12, or 13G10. Suchantibodies can include a heavy chain variable region that has at leasttwo, and usually all three, CDRs entirely or substantially from theheavy chain variable region of 15F7, 6C12, or 13G10 and/or a light chainvariable region having at least two, and usually all three, CDRsentirely or substantially from the light chain variable region of 15F7,6C12, or 13G10. The antibodies can include both heavy and light chains.A CDR is substantially from a corresponding 15F7, 6C12, or 13G10 CDRwhen it contains no more than 4, 3, 2, or 1 substitutions, insertions,or deletions, except that CDRH2 (when defined by Kabat) can have no morethan 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions. Suchantibodies can have at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identity to 15F7, 6C12, or 13G10 in the amino acid sequence of themature heavy and/or light chain variable regions and maintain theirfunctional properties, and/or differ from 15F7, 6C12, or 13G10 by asmall number of functionally inconsequential amino acid substitutions(e.g., conservative substitutions), deletions, or insertions.

B. Non-Human Antibodies

The production of other non-human antibodies, e.g., murine, guinea pig,primate, rabbit or rat, against the LG4-5 modules of the G domain oflaminin α4 can be accomplished by, for example, immunizing the animalwith laminin α4 or a fragment thereof. See Harlow & Lane, Antibodies, ALaboratory Manual (CSHP NY, 1988) (incorporated by reference for allpurposes). Such an immunogen can be obtained from a natural source, bypeptide synthesis, or by recombinant expression. Optionally, theimmunogen can be administered fused or otherwise complexed with acarrier protein. Optionally, the immunogen can be administered with anadjuvant. Several types of adjuvant can be used as described below.Complete Freund's adjuvant followed by incomplete adjuvant is preferredfor immunization of laboratory animals. Rabbits or guinea pigs aretypically used for making polyclonal antibodies. Mice are typically usedfor making monoclonal antibodies. Antibodies are screened for specificbinding to the LG4-5 modules of the G domain of laminin α4. Suchscreening can be accomplished by determining binding of an antibody to acollection of laminin α4 variants, such as laminin α4 variantscontaining the LG1-3 modules of the G domain, the LG1-5 modules of the Gdomain, and the LG4-5 modules of the G domain, and determining whichlaminin α4 variants bind to the antibody. Binding can be assessed, forexample, by Western blot, FACS or ELISA.

C. Humanized Antibodies

A humanized antibody is a genetically engineered antibody in which theCDRs from a non-human “donor” antibody are grafted into human “acceptor”antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and5,585,089; Winter, U.S. Pat. No. 5,225,539, Carter, U.S. Pat. No.6,407,213, Adair, U.S. Pat. No. 5,859,205 6,881,557, Foote, U.S. Pat.No. 6,881,557). The acceptor antibody sequences can be, for example, amature human antibody sequence, a composite of such sequences, aconsensus sequence of human antibody sequences, or a germline regionsequence. Thus, a humanized antibody is an antibody having some or allCDRs entirely or substantially from a donor antibody and variable regionframework sequences and constant regions, if present, entirely orsubstantially from human antibody sequences. Similarly a humanized heavychain has at least one, two and usually all three CDRs entirely orsubstantially from a donor antibody heavy chain, and a heavy chainvariable region framework sequence and heavy chain constant region, ifpresent, substantially from human heavy chain variable region frameworkand constant region sequences. Similarly a humanized light chain has atleast one, two and usually all three CDRs entirely or substantially froma donor antibody light chain, and a light chain variable regionframework sequence and light chain constant region, if present,substantially from human light chain variable region framework andconstant region sequences. Other than nanobodies and dAbs, a humanizedantibody comprises a humanized heavy chain and a humanized light chain.A CDR in a humanized antibody is substantially from a corresponding CDRin a non-human antibody when at least 85%, 90%, 95% or 100% ofcorresponding residues (as defined by Kabat) are identical between therespective CDRs. The variable region framework sequences of an antibodychain or the constant region of an antibody chain are substantially froma human variable region framework sequence or human constant regionrespectively when at least 85%, 90%, 95% or 100% of correspondingresidues defined by Kabat are identical.

Although humanized antibodies often incorporate all six CDRs (preferablyas defined by Kabat) from a mouse antibody, they can also be made withless than all CDRs (e.g., at least 3, 4, or 5 CDRs) from a mouseantibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos etal., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al.,Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology,164:1432-1441, 2000).

In some antibodies only part of the CDRs, namely the subset of CDRresidues required for binding, termed the SDRs, are needed to retainbinding in a humanized antibody. CDR residues not contacting antigen andnot in the SDRs can be identified based on previous studies (for exampleresidues H60-H65 in CDR H2 are often not required), from regions ofKabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol.Biol. 196:901, 1987), by molecular modeling and/or empirically, or asdescribed in Gonzales et al., Mol. Immunol. 41: 863, 2004. In suchhumanized antibodies at positions in which one or more donor CDRresidues is absent or in which an entire donor CDR is omitted, the aminoacid occupying the position can be an amino acid occupying thecorresponding position (by Kabat numbering) in the acceptor antibodysequence. The number of such substitutions of acceptor for donor aminoacids in the CDRs to include reflects a balance of competingconsiderations. Such substitutions are potentially advantageous indecreasing the number of mouse amino acids in a humanized antibody andconsequently decreasing potential immunogenicity. However, substitutionscan also cause changes of affinity, and significant reductions inaffinity are preferably avoided. Positions for substitution within CDRsand amino acids to substitute can also be selected empirically.

The human acceptor antibody sequences can optionally be selected fromamong the many known human antibody sequences to provide a high degreeof sequence identity (e.g., 65-85% identity) between a human acceptorsequence variable region frameworks and corresponding variable regionframeworks of a donor antibody chain.

Examples of acceptor sequences for the heavy chain are the human matureheavy chain variable regions with NCBI accession codes ACF36857.1 andBAC01530.1 (SEQ ID NOS:51 and 52, respectively). Other versions ofACF36857.1 and BAC01530.1 are SEQ ID NOS:96 and 97, respectively. Theseacceptor sequences include two CDRs having the same canonical form asmouse 15F7 heavy chain. Examples of acceptor sequences for the lightchain are the human mature light chain variable regions with NCBIaccession codes AAY33350.1 and BAC01583.1 (SEQ ID NOS:53 and 54,respectively). Another version of BAC01583.1 is SEQ ID NO:98. Theseacceptor sequences include three CDRs having the same canonical form asmouse 15F7 light chain.

Certain amino acids from the human variable region framework residuescan be selected for substitution based on their possible influence onCDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid can be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region or within a CDR as defined by        Chothia but not Kabat,    -   (3) otherwise interacts with a CDR region (e.g. is within about        6 Å of a CDR region), (e.g., identified by modeling the light or        heavy chain on the solved structure of a homologous known        immunoglobulin chain), or    -   (4) is a residue participating in the VL-VH interface.

Framework residues from classes (1) through (3) as defined by Queen,U.S. Pat. No. 5,530,101, are sometimes alternately referred to ascanonical and vernier residues. Framework residues that help define theconformation of a CDR loop are sometimes referred to as canonicalresidues (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Thornton &Martin, J. Mol. Biol. 263:800-815 (1996)). Framework residues thatsupport antigen-binding loop conformations and play a role infine-tuning the fit of an antibody to antigen are sometimes referred toas vernier residues (Foote & Winter, J. Mol. Viol 224:487-499 (1992)).

Other framework residues that are candidates for substitution areresidues creating a potential glycosylation site. Still other candidatesfor substitution are acceptor human framework amino acids that areunusual for a human immunoglobulin at that position. These amino acidscan be substituted with amino acids from the equivalent position of themouse donor antibody or from the equivalent positions of more typicalhuman immunoglobulins.

Exemplary humanized antibodies are humanized forms of the mouse 15F7antibody, designated Hu15F7. The mouse antibody comprises mature heavyand light chain variable regions having amino acid sequences comprisingSEQ ID NO:16 and SEQ ID NO:17, respectively. The invention provides twoexemplified humanized mature heavy chain variable regions: Hu15F7VHv1(H1; SEQ ID NO:57) and Hui5F7VHv2 (H2; SEQ ID NO:58). The inventionfurther provides two exemplified human mature light chain variableregions: Hui5F7VLv1 (L1; SEQ ID NO:59) and Hui5F7VLv2 (L2; SEQ IDNO:60).

For reasons such as possible influence on CDR conformation and/orbinding to antigen, mediating interaction between heavy and lightchains, interaction with the constant region, being a site for desiredor undesired post-translational modification, being an unusual residuefor its position in a human variable region sequence and thereforepotentially immunogenic, getting aggregation potential, and otherreasons, the following 20 variable region framework positions wereconsidered as candidates for substitutions in the two exemplified humanmature light chain variable regions and the two exemplified human matureheavy chain variable regions, as further specified in the examples: L8(P8S), L42 (K42N), L45 (K45R), L49 (Y49S), L69 (T69K), L80 (P80T), H1(Q1E), H20 (V20L), H27 (G27Y), H30 (S30T), H38 (R38K), H40 (A40R), H41(P41A), H48 (M48I), H66 (R66K), H67 (V67A), H69 (I69L), H75 (T75S), H82A(S(82A)R), and H91 (Y91F). In addition, L104 (L104V) was considered as acandidate for substitution in the two exemplified human mature lightchain variable regions.

Here, as elsewhere, the first-mentioned residue is the residue of ahumanized antibody formed by grafting Kabat CDRs into a human acceptorframework, and the second-mentioned residue is a residue beingconsidered for replacing such residue. Thus, within variable regionframeworks, the first mentioned residue is human, and within CDRs, thefirst mentioned residue is mouse.

Exemplified antibodies include any permutations or combinations of theexemplified mature heavy and light chain variable regions (e.g.,VHv1/VLv1 or H1L1, VHv1/VLv2 or H1L2, VHv2/VLv1 or H2L1, and VHv2/VLv2or H2L2). For example, the H1L1 antibody, which includes 14 heavy chainbackmutations or other mutations and 6 light chain backmutations asdescribed below, binds to laminin α4 and inhibits MCAM binding tolaminin α4 at a level that is substantially the same as, if not superiorto, a chimeric 19C12 antibody (see FIGS. 8-10). Comparable results areseen with the H1L2, H2L1, and H2L2 antibodies (see FIGS. 8-10).

The invention provides variants of the H1L1 humanized 15F7 antibody inwhich the humanized mature heavy chain variable region shows at least90%, 95%, 96%, 97%, 98%, or 99% identity to H1 (SEQ ID NO:57) and thehumanized mature light chain variable region shows at least 90%, 95%,96%, 97%, 98%, or 99% identity to L1 (SEQ ID NO:59). In some suchantibodies at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or all 20 of the backmutations or other mutations inH1L1 are retained. The invention also provides variants of the H2L1humanized 15F7 antibody in which the humanized mature heavy chainvariable region shows at least 90%, 95%, 96%, 97%, 98%, or 99% identityto H2 (SEQ ID NO:58) and the humanized mature light chain variableregion shows at least 90%, 95%, 96%, 97%, 98%, or 99% identity to L1(SEQ ID NO:59). In some such antibodies at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of the backmutations orother mutations in H2L1 are retained. In some antibodies, at least oneof positions H1, H20, H27, H30, H38, H40, H48, H66, H67, H75, H82A, andH91 in the Vh region is occupied by E, L, Y, T, K, R, I, K, A, S, R, andF, respectively. In some antibodies, positions H1, H20, H27, H30, H38,H40, H48, H66, H67, H75, H82A, and H91 in the Vh region are occupied byE, L, Y, T, K, R, I, K, A, S, R, and F, respectively, such as in versionH2. In some antibodies, at least one of positions H41 and H69 in the Vhregion is occupied by A and L, respectively. In some antibodies,positions H41 and H69 in the Vh region are occupied by A and L,respectively, such as in version H1. In some antibodies, at least one ofpositions L42, L49, and L69 in the Vk region is occupied by N, S, and K,respectively. In some antibodies, positions L42, L49, and L69 in the Vkregion are occupied by N, S, and K, respectively, such as in version L2.In some antibodies, at least one of positions L8, L45, and L80 in the Vkregion is occupied by S, R, and T, respectively. In some antibodies, atleast one of positions L8, L45, L80, and L104 in the Vk region isoccupied by S, R, T, and V, respectively. In some antibodies, positionsL8, L45, and L80 in the Vk region are occupied by S, R, and T,respectively, such as in version L1. In some antibodies, L104 isoccupied by V, such as in version L2. The CDR regions of such humanizedantibodies can be identical or substantially identical to the CDRregions of H1L1, which are the same as those of the mouse donorantibody. The CDR regions can be defined by any conventional definition(e.g., Chothia) but are preferably as defined by Kabat.

The invention also provides variants of the other exemplified Hu15F7antibodies. Such variants have mature light and heavy chain variableregions showing at least 90%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the mature light and heavy chain variable regions of theexemplified humanized 15F7 H1L2, H2L1, or H2L2 antibodies. The CDRregions of such humanized antibodies can be identical or substantiallyidentical to those of the mouse donor antibody. The CDR regions can bedefined by any conventional definition (e.g., Chothia) but arepreferably defined by Kabat. Variable regions framework positions are inaccordance with Kabat numbering unless otherwise stated. Other suchvariants typically differ from the sequences of the exemplified Hu15F7antibodies by a small number (e.g., typically no more than 1, 2, 3, 5,10, or 15) of replacements, deletions or insertions. Such differencesare usually in the framework but can also occur in the CDRs.

A possibility for additional variation in humanized 15F7 variants isadditional backmutations in the variable region frameworks. Many of theframework residues not in contact with the CDRs in the humanized mAb canaccommodate substitutions of amino acids from the correspondingpositions of the donor mouse mAb or other mouse or human antibodies, andeven many potential CDR-contact residues are also amenable tosubstitution. Even amino acids within the CDRs may be altered, forexample, with residues found at the corresponding position of the humanacceptor sequence used to supply variable region frameworks. Inaddition, alternate human acceptor sequences can be used, for example,for the heavy and/or light chain. If different acceptor sequences areused, one or more of the backmutations recommended above may not beperformed because the corresponding donor and acceptor residues arealready the same without backmutations.

Preferably, replacements or backmutations in Hu15F7 (whether or notconservative) have no substantial effect on the binding affinity orpotency of the humanized mAb, that is, its ability to bind to laminin α4or the LG4-5 modules of the G domain of laminin α4 (e.g., the potency insome or all of the assays described in the present examples of thevariant humanized 15F7 antibody is essentially the same, i.e., withinexperimental error, as that of murine 15F7 or H1L1).

D. Chimeric and Veneered Antibodies

The invention further provides chimeric and veneered forms of non-humanantibodies, particularly the 15F7, 6C12, or 13G10 antibodies of theexamples.

A chimeric antibody is an antibody in which the mature variable regionsof light and heavy chains of a non-human antibody (e.g., a mouse) arecombined with human light and heavy chain constant regions. Suchantibodies substantially or entirely retain the binding specificity ofthe mouse antibody, and are about two-thirds human sequence.

A veneered antibody is a type of humanized antibody that retains someand usually all of the CDRs and some of the non-human variable regionframework residues of a non-human antibody but replaces other variableregion framework residues that may contribute to B- or T-cell epitopes,for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) withresidues from the corresponding positions of a human antibody sequence.The result is an antibody in which the CDRs are entirely orsubstantially from a non-human antibody and the variable regionframeworks of the non-human antibody are made more human-like by thesubstitutions. Veneered forms of the 15F7 antibody are included in theinvention.

E. Human Antibodies

Human antibodies against the LG4-5 modules of the G domain of laminin α4are provided by a variety of techniques described below. Some humanantibodies are selected by competitive binding experiments, by the phagedisplay method of Winter, above, or otherwise, to have the same epitopespecificity as a particular mouse antibody, such as one of the mousemonoclonal antibodies described in the examples. Human antibodies canalso be screened for a particular epitope specificity by using only afragment of laminin α4, such as a laminin α4 variant containing only theLG4-5 modules of the G domain, as the target antigen, and/or byscreening antibodies against a collection of laminin α4 variants, suchas laminin α4 variants containing the LG1-3 modules of the G domain, theLG1-5 modules of the G domain, and the LG4-5 modules of the G domain.

Methods for producing human antibodies include the trioma method ofOestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No.4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, use oftransgenic mice including human immunoglobulin genes (see, e.g., Lonberget al., WO93/12227 (1993); U.S. Pat. No. 5,877,397, U.S. Pat. No.5,874,299, U.S. Pat. No. 5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat.No. 5,770,429, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,633,425, U.S.Pat. No. 5,625,126, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,545,806,Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996),Kucherlapati, WO 91/10741 (1991)) and phage display methods (see, e.g.,Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, U.S. Pat.No. 5,877,218, U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,858,657, U.S.Pat. No. 5,837,242, U.S. Pat. No. 5,733,743 and U.S. Pat. No.5,565,332).

F. Selection of Constant Region

The heavy and light chain variable regions of chimeric, humanized(including veneered), or human antibodies can be linked to at least aportion of a human constant region. The choice of constant regiondepends, in part, on whether antibody-dependent complement and/orcellular mediated cytotoxicity is desired. For example, human isotypesIgG1 and IgG3 have complement-mediated cytotoxicity and human isotypesIgG2 and IgG4 do not. Human IgG1 and IgG3 also induce stronger cellmediated effector functions than human IgG2 and IgG4. A human IgG1constant region suitable for inclusion in the antibodies can have thesequence of SEQ ID NO:61. The C-terminal lysine of SEQ ID NO:61 can beomitted, in which case the IgG1 constant region has the amino acidsequence of SEQ ID NO:91. Light chain constant regions can be lambda orkappa. A human kappa light chain constant region suitable for inclusionin the antibodies can have the sequence of SEQ ID NO:90. SEQ ID NO:90can be encoded by the nucleic acid sequence of SEQ ID NO:93. TheN-terminal arginine of SEQ ID NO:90 can be omitted, in which case thekappa light chain constant region has the amino acid sequence of SEQ IDNO:62. SEQ ID NO:62 can be encoded by the nucleic acid sequence of SEQID NO:102. Antibodies can be expressed as tetramers containing two lightand two heavy chains, as separate heavy chains, light chains, as Fab,Fab′, F(ab′)2, and Fv, or as single chain antibodies in which heavy andlight chain variable domains are linked through a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype bind to a non-polymorphic region of a one or more otherisotypes. Thus, for example, another heavy chain constant region is ofthe IgG1 G1m3 allotype and has the amino acid sequence of SEQ ID NO:89.SEQ ID NO:89 can be encoded by the nucleic acid sequence of SEQ IDNO:92. Another heavy chain constant region of the IgG1 G1m3 allotype hasthe amino acid sequence of SEQ ID NO:101. Reference to a human constantregion includes a constant region with any natural allotype or anypermutation of residues occupying polymorphic positions in naturalallotypes.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position250 and/or a Leu at position 428 (EU numbering) for increasing thehalf-life of an antibody. Substitution at any of positions 234, 235, 236and/or 237 reduces affinity for Fcγ receptors, particularly FcγRIreceptor (see, e.g., U.S. Pat. No. 6,624,821). An alanine substitutionat positions 234, 235, and 237 of human IgG1 can be used for reducingeffector functions. Optionally, positions 234, 236 and/or 237 in humanIgG2 are substituted with alanine and position 235 with glutamine. See,e.g., U.S. Pat. No. 5,624,821. In some antibodies, a mutation at one ormore of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 by EUnumbering of human IgG1 is used. In some antibodies, a mutation at oneor more of positions 318, 320, and 322 by EU numbering of human IgG1 isused. In some antibodies, positions 234 and/or 235 are substituted withalanine and/or position 329 is substituted with glycine. In someantibodies, positions 234 and 235 are substituted with alanine, such asin SEQ ID NO:101. In some antibodies, the isotype is human IgG2 or IgG4.

G. Expression of Recombinant Antibodies

A number of methods are known for producing chimeric and humanizedantibodies using an antibody-expressing cell line (e.g., hybridoma). Forexample, the immunoglobulin variable regions of antibodies can be clonedand sequenced using well known methods. In one method, the heavy chainvariable VH region is cloned by RT-PCR using mRNA prepared fromhybridoma cells. Consensus primers are employed to the VH region leaderpeptide encompassing the translation initiation codon as the 5′ primerand a g2b constant regions specific 3′ primer. Exemplary primers aredescribed in U.S. patent publication US 2005/0009150 by Schenk et al.(hereinafter “Schenk”). The sequences from multiple, independentlyderived clones can be compared to ensure no changes are introducedduring amplification. The sequence of the VH region can also bedetermined or confirmed by sequencing a VH fragment obtained by 5′ RACERT-PCR methodology and the 3′ g2b specific primer.

The light chain variable VL region can be cloned in an analogous manner.In one approach, a consensus primer set is designed for amplification ofVL regions using a 5′ primer designed to hybridize to the VL regionencompassing the translation initiation codon and a 3′ primer specificfor the Ck region downstream of the V-J joining region. In a secondapproach, 5′RACE RT-PCR methodology is employed to clone a VL encodingcDNA. Exemplary primers are described in Schenk, supra. The clonedsequences are then combined with sequences encoding human (or othernon-human species) constant regions. Exemplary sequences encoding humanconstant regions include SEQ ID NO:61, which encodes a human IgG1constant region, and SEQ ID NO:62, which encodes a human kappa lightchain constant region.

In one approach, the heavy and light chain variable regions arere-engineered to encode splice donor sequences downstream of therespective VDJ or VJ junctions and are cloned into a mammalianexpression vector, such as pCMV-hγ1 for the heavy chain and pCMV-Mcl forthe light chain. These vectors encode human γ1 and Ck constant regionsas exonic fragments downstream of the inserted variable region cassette.Following sequence verification, the heavy chain and light chainexpression vectors can be co-transfected into CHO cells to producechimeric antibodies. Conditioned media is collected 48 hourspost-transfection and assayed by western blot analysis for antibodyproduction or ELISA for antigen binding. The chimeric antibodies arehumanized as described above.

Chimeric, veneered, humanized, and human antibodies are typicallyproduced by recombinant expression. Recombinant polynucleotideconstructs typically include an expression control sequence operablylinked to the coding sequences of antibody chains, including naturallyassociated or heterologous expression control elements, such as apromoter. The expression control sequences can be promoter systems invectors capable of transforming or transfecting eukaryotic orprokaryotic host cells. Once the vector has been incorporated into theappropriate host, the host is maintained under conditions suitable forhigh level expression of the nucleotide sequences and the collection andpurification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers, e.g., ampicillinresistance or hygromycin resistance, to permit detection of those cellstransformed with the desired DNA sequences.

E. coli is one prokaryotic host useful for expressing antibodies,particularly antibody fragments. Microbes, such as yeast, are alsouseful for expression. Saccharomyces is a yeast host with suitablevectors having expression control sequences, an origin of replication,termination sequences, and the like as desired. Typical promotersinclude 3-phosphoglycerate kinase and other glycolytic enzymes.Inducible yeast promoters include, among others, promoters from alcoholdehydrogenase, isocytochrome C, and enzymes responsible for maltose andgalactose utilization.

Mammalian cells can be used for expressing nucleotide segments encodingimmunoglobulins or fragments thereof. See Winnacker, From Genes toClones, (VCH Publishers, N Y, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped, and include CHO cell lines, various COS cell lines, HeLacells, HEK293 cells, L cells, and non-antibody-producing myelomasincluding Sp2/0 and NSO. The cells can be nonhuman. Expression vectorsfor these cells can include expression control sequences, such as anorigin of replication, a promoter, an enhancer (Queen et al., Immunol.Rev. 89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Expression control sequences caninclude promoters derived from endogenous genes, cytomegalovirus, SV40,adenovirus, bovine papillomavirus, and the like. See Co et al., J.Immunol. 148:1149 (1992).

Alternatively, antibody coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,U.S. Pat. No. 5,741,957, U.S. Pat. No. 5,304,489, U.S. Pat. No.5,849,992). Suitable transgenes include coding sequences for lightand/or heavy chains operably linked with a promoter and enhancer from amammary gland specific gene, such as casein or beta lactoglobulin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by methods depending on the type of cellular host.For example, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment, electroporation,lipofection, biolistics, or viral-based transfection can be used forother cellular hosts. Other methods used to transform mammalian cellsinclude the use of polybrene, protoplast fusion, liposomes,electroporation, and microinjection. For production of transgenicanimals, transgenes can be microinjected into fertilized oocytes or canbe incorporated into the genome of embryonic stem cells, and the nucleiof such cells transferred into enucleated oocytes.

Having introduced vector(s) encoding antibody heavy and light chainsinto cell culture, cell pools can be screened for growth productivityand product quality in serum-free media. Top-producing cell pools canthen be subjected of FACS-based single-cell cloning to generatemonoclonal lines. Specific productivities above 50 pg or 100 pg per cellper day, which correspond to product titers of greater than 7.5 g/Lculture, can be used. Antibodies produced by single cell clones can alsobe tested for turbidity, filtration properties, PAGE, IEF, UV scan,HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, andbinding assay, such as ELISA or Biacore. A selected clone can then bebanked in multiple vials and stored frozen for subsequent use.

Once expressed, antibodies can be purified according to standardprocedures of the art, including protein A capture, HPLC purification,column chromatography, gel electrophoresis and the like (see generally,Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

Methodology for commercial production of antibodies can be employed,including codon optimization, selection of promoters, selection oftranscription elements, selection of terminators, serum-free single cellcloning, cell banking, use of selection markers for amplification ofcopy number, CHO terminator, or improvement of protein titers (see,e.g., U.S. Pat. No. 5,786,464, U.S. Pat. No. 6,114,148, U.S. Pat. No.6,063,598, U.S. Pat. No. 7,569,339, WO2004/050884, WO2008/012142,WO2008/012142, WO2005/019442, WO2008/107388, and WO2009/027471, and U.S.Pat. No. 5,888,809).

H. Nucleic Acids

The invention further provides nucleic acids encoding any of the heavyand light chains described above (e.g., SEQ ID NOS:63-64, 67-68, 71-73,and 77-80). SEQ ID NOS:92-93, 99-100, and 102 are additional examples ofnucleic acids encoding heavy and light chains described above.Typically, the nucleic acids also encode a signal peptide fused to themature heavy and light chains (e.g., signal peptides having amino acidsequences of SEQ ID NOS:18, 28, 39, and 40 (heavy chain) and 19, 29, and41 (light chain) that can be encoded by SEQ ID NOS:65, 69, 74, and 75,respectively (heavy chain) and 66, 70, and 76, respectively (lightchain)). Coding sequences of nucleic acids can be operably linked withregulatory sequences to ensure expression of the coding sequences, suchas a promoter, enhancer, ribosome binding site, transcriptiontermination signal, and the like. The nucleic acids encoding heavy andlight chains can occur in isolated form or can be cloned into one ormore vectors. The nucleic acids can be synthesized by, for example,solid state synthesis or PCR of overlapping oligonucleotides. Nucleicacids encoding heavy and light chains can be joined as one contiguousnucleic acid, e.g., within an expression vector, or can be separate,e.g., each cloned into its own expression vector.

I. Conjugated Antibodies

Antibodies that specifically bind to antigens, such as the LG4-5 modulesof the G domain of laminin α4, that are preferentially present incancers and tumors are useful in targeting cancer or tumor cells fordestruction. Likewise, antibodies that specifically bind the LG4-5modules of the G domain of laminin α4 can be useful in targeting cellsinvolved in autoimmune diseases or any other diseases mediated at leastin part by expression of the LG4-5 modules of the G domain of lamininα4. For example, such antibodies can be conjugated with othertherapeutic or cytotoxic agents. See WO 03/057838. Likewise, suchantibodies can be conjugated with other proteins, other antibodies,and/or detectable labels. See WO 03/057838; U.S. Pat. No. 8,455,622.Such therapeutic agents can be any agent that can be used to treat,combat, ameliorate, prevent, or improve an unwanted condition or diseasein a patient. Examples of such conditions or diseases include cancersand autoimmune diseases. Therapeutic agents include cytotoxic agents,cytostatic agents, radiotherapeutic agents, immunomodulators, or anybiologically active agents that facilitate or enhance the activity ofthe antibody. Such cytotoxic agents can be any agent that is toxic to acell. A cytostatic agent can be any agent that inhibits cellproliferation. An immunomodulator can be any agent that stimulates orinhibits the development or maintenance of an immunologic response. Aradiotherapeutic agent can be any molecule or compound that emitsradiation. If such therapeutic or cytotoxic agents are coupled to atumor-specific antibody, such as the antibodies described herein, thecoupled therapeutic or cytotoxic agents will have a specific affinityfor tumor cells or cancer cells over normal cells. Likewise, the coupledtherapeutic or cytotoxic agents will have a specific affinity forlaminin-α4-expressing cells over other cells. Consequently,administration of the conjugated antibodies directly targets cancercells with minimal damage to surrounding normal, healthy tissue. Thiscan be particularly useful for therapeutic or cytotoxic agents that aretoo toxic to be administered on their own. In addition, smallerquantities of the therapeutic or cytotoxic agents can be used.

Antibodies can be modified to act as immunotoxins. See, e.g., U.S. Pat.No. 5,194,594. For example, ricin, a cellular toxin derived from plants,can be coupled to antibodies by using the bifunctional reagentsS-acetylmercaptosuccinic anhydride for the antibody and succinimidyl3-(2-pyridyldithio)propionate for ricin. See Pietersz et al., CancerRes. 48(16):4469-4476 (1998). The coupling results in loss of B-chainbinding activity of ricin, while impairing neither the toxic potentialof the A-chain of ricin nor the activity of the antibody. Similarly,saporin, an inhibitor of ribosomal assembly, can be coupled toantibodies via a disulfide bond between chemically inserted sulfhydrylgroups. See Polito et al., Leukemia 18:1215-1222 (2004).

Radioisotopes can also be linked to antibodies, such as, for example,yttrium⁹⁰ (90Y), indium¹¹¹ (111In), ¹³¹I, ⁹⁹mTc, radiosilver-111,radiosilver-199, and Bismuth²¹³. Linkage of radioisotopes to antibodiesmay be performed with conventional bifunction chelates. Forradiosilver-11 and radiosilver-199 linkage, sulfur-based linkers may beused. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linkage ofsilver radioisotopes may involve reducing the immunoglobulin withascorbic acid. For radioisotopes such as 111In and 90Y, ibritumomabtiuxetan can be used and will react with such isotopes to form111In-ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively.See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

Other therapeutic agents may also be linked to antibodies. Therapeuticagents are usually cytotoxic or cytostatic. For example, antibodies canbe conjugated with toxic chemotherapeutic drugs such as maytansine,geldanamycin, tubulin inhibitors, such as tubulin binding agents (e.g.,auristatins), or minor groove binding agents, such as calicheamicin.Other representative therapeutic agents include agents known to beuseful for treatment, management, or amelioration of a cancer or anautoimmune disease or symptoms of a cancer or an autoimmune disease.Examples of such therapeutic agents are disclosed elsewhere herein.

Antibodies can also be coupled with other proteins. For example,antibodies can be coupled with Fynomers. Fynomers are small bindingproteins (e.g., 7 kDa) derived from the human Fyn SH3 domain. They canbe stable and soluble, and they can lack cysteine residues and disulfidebonds. Fynomers can be engineered to bind to target molecules with thesame affinity and specificity as antibodies. They are suitable forcreating multi-specific fusion proteins based on antibodies. Forexample, Fynomers can be fused to N-terminal and/or C-terminal ends ofantibodies to create bi- and tri-specific FynomAbs with differentarchitectures. Fynomers can be selected using Fynomer libraries throughscreening technologies using FACS, Biacore, and cell-based assays thatallow efficient selection of Fynomers with optimal properties. Examplesof Fynomers are disclosed in Grabulovski et al., J. Biol. Chem.282:3196-3204 (2007); Bertschinger et al., Protein Eng. Des. Sel.20:57-68 (2007); Schlatter et al., MAbs. 4:497-508 (2011); Banner etal., Acta. Crystallogr. D. Biol. Crystallogr. 69(Pt6):1124-1137 (2013);and Brack et al., Mol. Cancer Ther. 13:2030-2039 (2014).

The antibodies disclosed herein can also be coupled or conjugated to oneor more other antibodies (e.g., to form antibody heteroconjugates). Suchother antibodies can bind to different epitopes within the LG4-5 modulesof the G domain of laminin α4, or can bind to a different targetantigen.

Antibodies can also be coupled with a detectable label. Such antibodiescan be used, for example, for diagnosing a cancer or an autoimmunedisease, for monitoring progression of a cancer or an autoimmunedisease, and/or for assessing efficacy of treatment. Such antibodies canbe useful for performing such determinations in subjects having or beingsusceptible to a cancer or an autoimmune disease, or in appropriatebiological samples obtained from such subjects. Representativedetectable labels that may be coupled or linked to an antibody includevarious enzymes, such as horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic groups, suchstreptavidin/biotin and avidin/biotin; fluorescent materials, such asumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as luminol; bioluminescent materials, suchas luciferase, luciferin, and aequorin; radioactive materials, such asradiosilver-111, radiosilver-199, Bismuth²¹³, iodine (¹³¹I, ¹²⁵I, ¹²³I,¹²¹I), carbon (¹⁴C), sulfur (⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In,¹¹²In, ¹¹¹In), technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga,⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine(¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc,¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd,¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³5 n, and ¹¹⁷Tin; positron emitting metalsusing various positron emission tomographies; nonradioactiveparamagnetic metal ions; and molecules that are radiolabelled orconjugated to specific radioisotopes.

Therapeutic agents, other proteins, other antibodies, and/or detectablelabels may be coupled or conjugated, directly or indirectly through anintermediate (e.g., a linker), to a murine, chimeric, veneered, orhumanized antibody using techniques known in the art. See e.g., Arnon etal., “Monoclonal Antibodies For Immunotargeting Of Drugs In CancerTherapy,” in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.(eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,“Antibodies For Drug Delivery,” in Controlled Drug Delivery (2nd Ed.),Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,“Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” inMonoclonal Antibodies 84: Biological And Clinical Applications, Pincheraet al. (eds.), pp. 475-506 (1985); “Analysis, Results, And FutureProspective Of The Therapeutic Use Of Radiolabeled Antibody In CancerTherapy,” in Monoclonal Antibodies For Cancer Detection And Therapy,Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe etal., Immunol. Rev., 62:119-58 (1982). Suitable linkers include, forexample, cleavable and non-cleavable linkers. Different linkers thatrelease the drugs under acidic or reducing conditions or on exposure tospecific proteases can be employed. Likewise, different linkers thatrelease the coupled therapeutic agents, proteins, antibodies, and/ordetectable labels under acidic or reducing conditions, on exposure tospecific proteases, or under other defined conditions can be employed.

V. Therapeutic Applications

The above antibodies can be used for treating or effecting prophylaxisof a disease in a patient having or at risk for the disease mediated atleast in part by expression of the LG4-5 modules of the G domain oflaminin α4. In some such diseases, the LG4-5 modules of the G domain oflaminin α4 contribute to progression of the disease by means ofinteraction with a heparan sulfate proteoglycan, such as syndecan-1,syndecan-2, syndecan-3, or syndecan-4.

Such a disease can be a cancer. Exemplary cancers to be treated havebeen described above. The methods are particularly amendable totreatment of a cancer showing a detectable level of the LG4-5 modules ofthe G domain of laminin α4 (e.g., by immunoassay using one of thedisclosed antibodies). Some such cancers show an increased expression oflaminin α4 compared with noncancerous tissue of the same type. Some suchcancers are those that have been reported to be associated with lamininα4 expression, including glioma and glial tumors (see, e.g., Nagato etal., Int. J. Cancer 117:41-50 (2005); Ljubimova et al., Cancer101(3):604-612 (2004)), hepatocellular carcinoma (see, e.g., Huang etal., J Cancer Res. Clin. Oncol. 134(6):705-14 (2008)), renal cellcarcinoma (see, e.g., Vainionpaa et al., Lab Invest. 87(8):780-791(2007)), oral squamous cell carcinoma (see, e.g., Takkunen et al.,Histochem. Cell Biol. 130(3):509-525 (2008)), prostate cancer (see,e.g., Sprenger et al., Neoplasia 10(12):1350-1361 (2008)), and melanoma(see, e.g., Lugassy et al., J. Cutan. Pathol. 36(12):1237-1243 (2009)).Some such cancers are those associated with expression of one or moresyndecans, such as breast cancer, cancer of the uterine cervix,colorectal cancer, endometrial cancer, fibrosarcoma, gallbladder cancer,gastric cancer, glioma, head and neck cancer, hepatocellular carcinoma,juvenile nasopharyngeal angiofibroma, lung cancer, melanoma,mesothelioma, myeloma, neuroendocrine tumors, oral cancer, oral squamouscell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, andprostate cancer. See, e.g., Theocharis et al., FEBS Journal277:3904-3923 (2010). Some of the above antibodies are useful for thetreatment of melanoma, breast cancer, lung cancer, and colorectalcancer. Some antibodies may be useful for the treatment of uterinecancer, cervical cancer, endometrial cancer, fibrosarcoma, gallbladdercancer, gastric cancer, glioma, head and neck cancer, hepatocelleularcarcinoma, juvenile nasopharyngeal angiofibroma, mesothelioma, myeloma,neuroendocrine, tumors, oral cancer, squamous cell carcinoma, oralsquamous cell carcinoma, osteosarcoma, pancreatic cancer and/or prostatecancer. Some antibodies may be useful in treating metastatic tumors. Insome instances the patient has a brain cancer or another type of CNS orintracranial tumor. For example, the patient can have an astrocytictumor (e.g., astrocytoma, anaplastic astrocytoma, glioblastoma,pilocytic astrocytoma, subependymal giant cell astrocytoma, pleomorphicxanthoastrocytoma), oligodendroglial tumor (e.g., oligodendroglioma,anaplastic oligodendroglioma), ependymal cell tumor (e.g., ependymoma,anaplastic ependymoma, myxopapillary ependymoma, subependymoma), mixedglioma (e.g., mixed oligoastrocytoma, anaplastic oligoastrocytoma),neuroepithelial tumor of uncertain origin (e.g., polar spongioblastoma,astroblastoma, gliomatosis cerebri), tumor of the choroid plexus (e.g.,choroid plexus papilloma, choroid plexus carcinoma), neuronal or mixedneuronal-glial tumor (e.g., gangliocytoma, dyplastic gangliocytoma ofcerebellum, ganglioglioma, anaplastic ganglioglioma, desmoplasticinfantile ganglioma, central neurocytoma, dysembryoplasticneuroepithelial tumor, olfactory neuroblastoma), pineal parenchyma tumor(e.g., pineocytoma, pineoblastoma, mixed pineocytoma/pineoblastoma), ortumor with mixed neuroblastic or glioblastic elements (e.g.,medulloepithelioma, medulloblastoma, neuroblastoma, retinoblastoma,ependymoblastoma). Some antibodies may be useful in treating othercancers.

Such a disease can be an autoimmune disease. Autoimmune diseases includesystemic autoimmune diseases, organ- or tissue-specific autoimmunediseases, and diseases that exhibit autoimmune-type expressions. Inthese diseases, the body develops a cellular and/or humoral immuneresponse against one of its own antigens, leading to destruction of thatantigen and potentially crippling and/or fatal consequences. Thecellular response if present can be B-cell or T-cell or both. TH17cells, a lineage T helper cells characterized by production ofinterleukin (IL)-17 and IL-22, have been reported to enter tissues tofacilitate pathogenic autoimmune responses, including multiple sclerosisin humans and experimental autoimmune encephalomyelitis (EAE) in mice.See, e.g., Cua et al., Nature 421: 744-748 (2003); Ivonov et al., Cell126: 1121-1133 (2006). TH17 cells may initiate or propagate aninflammatory response by their specific recruitment to and infiltrationof tissue.

Examples of autoimmune diseases include Graves' disease, Hashimoto'sthyroiditis, autoimmune polyglandular syndrome, insulin-dependentdiabetes mellitus (type 1 diabetes), insulin-resistant diabetes mellitus(type 2 diabetes), immune-mediated infertility, autoimmune Addison'sdisease, pemphigus vulgaris, pemphigus foliaceus, dermatitisherpetiformis, autoimmune alopecia, vitiligo, autoimmune hemolyticanemia, idiopathic thrombocytopenic purpura, autoimmune thrombocytopenicpurpura, pernicious anemia, myasthenia gravis, Guillain-Barre syndrome,stiff man syndrome, acute rheumatic fever, sympathetic ophthalmia,Goodpasture's syndrome, autoimmune uveitis, temporal arteritis, Bechet'sdisease, inflammatory bowel diseases, Crohn's disease, ulcerativecolitis, primary biliary cirrhosis, autoimmune hepatitis, autoimmuneoophoritis, fibromyalgia, polymyositis, dermatomyostis, ankylosingspondylitis, Takayashu arteritis, panniculitis, pemphigoid, vasculitisof unknown origin, anca negative vasculitis, anca positive vasculitis,systemic lupus erythematosus, psoriatic arthritis, rheumatoid arthritis,scleroderma, systemic necrotizing vasculitis, Wegener's granulomatosis,CREST syndrome, antiphospholipid syndrome, Sjogren's syndrome,eosinophilic gastroenteritis, atypical topical dermatitis,cardiomyopathy, post-infectious syndromes, postinfectiousendomyocarditis, celiac disease, multiple sclerosis, sarcoidosis, andpsoriasis

Although an understanding of mechanism is not required for practice, itis believed that any or all of the following mechanisms may contributeto treatment of cancer. The antibodies may treat the cancer byinhibiting tumor cell adhesion, inhibiting laminin-α4-mediated signalingevents, or inhibiting interaction of laminin α4 with a syndecan. Theantibodies may additionally or alternatively treat cancer by inducingprocessing or clearance of the LG4-5 modules of the G domain of lamininα4. The antibodies may additionally or alternatively inhibit metastasisor cancer cell invasion. Binding of antibodies to LG4-5 modules of the Gdomain of laminin α4 may also affect cell adhesion, signaling mechanismsinvolved in cell proliferation, growth, resisting cell death,angiogenesis, or other characteristics of cancers. The antibodies mayinhibit tumor growth via inhibiting Akt activation and subsequent cellsurvival/proliferation signaling. In some instances, the antibodiesdisrupt or inhibit angiogenesis by altering endothelial Dll4/Notchsignaling. In some cases, the disruption or inhibition of angiogenesisby the antibodies involves disrupting the interaction between laminin α4and integrins, such as integrins comprising integrin α2, integrin α6, orintegrin β1. Antibody-drug conjugates have additional mechanisms ofaction including the cytotoxic or cytostatic effect of the linked agent,typically after uptake within a cancer cell. Antibody-drug conjugatescan also act by such mechanisms of action in other targeted cells.Antibody-drug conjugates may also induce tumor-associated macrophagetoxicity.

Other diseases treatable by antibodies of the invention include obesityand obesity-related diseases, such as obesity-related orphan diseases.Obesity is a disease caused by excessive food energy intake, lack ofphysical activity, and/or genetic susceptibility. A body mass index(BMI) >35 indicates severe obesity, a BMI >40 indicates morbid obesity,and a BMI >45 indicates super obesity. Obesity-related diseases includediseases and disorders that are associated with, are caused by, orresult from obesity. Examples of obesity-related diseases includecardiovascular diseases, type 2 diabetes, sleep apnea, cancer,osteoarthritis, asthma, fatty liver, and non-alcoholic steatohepatitis(NASH).

NASH is characterized by hepatic inflammation and fat accumulation. Theprimary risk factors are obesity, diabetes, and dyslipidemia. There is astrong link with cirrhosis and hepatocarcinoma. NASH is associated withelevated AST/ALT (ratio of concentration of aspartate transaminase (AST)and alanine transaminase (ALT)), often without symptoms. Treatments forNASH include lifestyle changes (diet and exercise), bariatric surgery,and pharmaceuticals with mechanisms including absorption reduction(Xenical/Alli (lipase inhibitor)), appetite suppression (Belviq, Byetta,Symlin, Qsymia), and metabolic stimulation (Beloranib).

Examples of obesity-related orphan diseases include Prader-Willisyndrome (e.g., with hyperphagia), craniopharyngioma (e.g., withhyperphagia), Bardet-Biedl syndrome, Cohen syndrome, and MOMO syndrome.Prader-Willi syndrome is a rare genetic disease caused by genedeletion/silencing on chromosome 15. The symptoms include neurocognitivesymptoms (intellectual disability, autistic behaviors, uncontrolledappetite (hypothalamic)), slow metabolism, and endocrine disorders(e.g., growth hormone deficiency (GHD), adrenal deficiency (AD)).

Antibodies are administered in an effective regime meaning a dosage,route of administration and frequency of administration that delays theonset, reduces the severity, inhibits further deterioration, and/orameliorates at least one sign or symptom of a disorder being treated(e.g., cancer). If a patient is already suffering from a disorder, theregime can be referred to as a therapeutically effective regime. If thepatient is at elevated risk of the disorder relative to the generalpopulation but is not yet experiencing symptoms, the regime can bereferred to as a prophylactically effective regime. In some instances,therapeutic or prophylactic efficacy can be observed in an individualpatient relative to historical controls or past experience in the samepatient. In other instances, therapeutic or prophylactic efficacy can bedemonstrated in a preclinical or clinical trial in a population oftreated patients relative to a control population of untreated patients.

Exemplary dosages for an antibody are 0.1-20, or 0.5-5 mg/kg body weight(e.g., 0.5, 1, 2, 3, 4 or 5 mg/kg) or 10-1500 mg as a fixed dosage. Thedosage depends on the condition of the patient and response to priortreatment, if any, whether the treatment is prophylactic or therapeuticand whether the disorder is acute or chronic, among other factors.

Administration can be parenteral, intravenous, oral, subcutaneous,intra-arterial, intracranial, intrathecal, intraperitoneal, topical,intranasal or intramuscular. Some antibodies can be administered intothe systemic circulation by intravenous or subcutaneous administration.Intravenous administration can be, for example, by infusion over aperiod such as 30-90 min.

The frequency of administration depends on the half-life of the antibodyin the circulation, the condition of the patient and the route ofadministration among other factors. The frequency can be daily, weekly,monthly, quarterly, or at irregular intervals in response to changes inthe patient's condition or progression of the disorder being treated. Anexemplary frequency for intravenous administration is between weekly andquarterly over a continuous cause of treatment, although more or lessfrequent dosing is also possible. For subcutaneous administration, anexemplary dosing frequency is daily to monthly, although more or lessfrequent dosing is also possible.

The number of dosages administered depends on whether the disorder isacute or chronic and the response of the disorder to the treatment. Foracute disorders or acute exacerbations of a chronic disorder, between 1and 10 doses are often sufficient. Sometimes a single bolus dose,optionally in divided form, is sufficient for an acute disorder or acuteexacerbation of a chronic disorder. Treatment can be repeated forrecurrence of an acute disorder or acute exacerbation. For chronicdisorders, an antibody can be administered at regular intervals, e.g.,weekly, fortnightly, monthly, quarterly, every six months for at least1, 5 or 10 years, or the life of the patient.

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologically andpharmaceutically acceptable carriers, diluents, excipients orauxiliaries. The formulation depends on the route of administrationchosen. For injection, antibodies can be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHank's solution, Ringer's solution, or physiological saline or acetatebuffer (to reduce discomfort at the site of injection). The solution cancontain formulatory agents such as suspending, stabilizing and/ordispersing agents. Alternatively antibodies can be in lyophilized formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

Treatment with antibodies described herein can be combined with othertreatments effective against the disorder being treated. When used intreating cancer, the antibodies can be combined with chemotherapy,radiation, stem cell treatment, surgery, or treatment with otherbiologics including Herceptin® (trastuzumab) against the HER2 antigen,Avastin® (bevacizumab) against VEGF, or antibodies to the EGF receptor,such as (Erbitux®, cetuximab), and Vectibix® (panitumumab). Chemotherapyagents include chlorambucil, cyclophosphamide or melphalan,carboplatinum, daunorubicin, doxorubicin, idarubicin, and mitoxantrone,methotrexate, fludarabine, and cytarabine, etoposide or topotecan,vincristine and vinblastine.

VI. Other Applications

The antibodies can be used for detecting laminin α4 in the context ofclinical diagnosis or treatment or in research. More specifically, theantibodies can also be used for detecting the LG4-5 modules of the Gdomain of laminin α4, or fragments thereof, in the context of clinicaldiagnosis or treatment or in research. For example, the antibodies canbe used to detect the presence of the LG4-5 modules of the G domain oflaminin α4 in a biological sample as an indication that the biologicalsample comprises cancer cells or tumor cells. Binding of the antibodiesto the biological sample can be compared to binding of the antibodies toa control sample. The control sample and the biological sample cancomprise cells of the same tissue origin. In some assays, the cancercells that may be present in the biological sample arose from the samecell type as the type of cells in the control sample. Control samplesand biological samples can be obtained from the same individual ordifferent individuals and on the same occasion or on differentoccasions. If desired, multiple biological samples and multiple controlsamples are evaluated on multiple occasions to protect against randomvariation independent of the differences between the samples. A directcomparison can then be made between the biological sample(s) and thecontrol sample(s) to determine whether antibody binding (i.e., thepresence of the LG4-5 modules of the G domain of laminin α4) to thebiological sample(s) is increased, decreased, or the same relative toantibody binding to the control sample(s). Increased binding of theantibody to the biological sample(s) relative to the control sample(s)indicates the presence of cancer in the biological sample(s). In someinstances, the increased antibody binding is statistically significant.Optionally, antibody binding to the biological sample is at least1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 100-foldhigher than antibody binding to the control sample.

In addition, the antibodies can be used to detect the presence of theLG4-5 modules of the G domain of laminin α4 in a biological sample tomonitor and evaluate the efficacy of a therapeutic agent being used totreat a patient diagnosed with cancer. A biological sample from apatient diagnosed with cancer is evaluated to establish a baseline forthe binding of the antibodies to the sample (i.e., a baseline for thepresence of the LG4-5 modules of the G domain of laminin α4 in thesample) before commencing therapy with the therapeutic agent. In someinstances, multiple biological samples from the patient are evaluated onmultiple occasions to establish both a baseline and measure of randomvariation independent of treatment. A therapeutic agent is thenadministered in a regime. The regime may include multipleadministrations of the agent over a period of time. Optionally, bindingof the antibodies (i.e., presence of the LG4-5 modules of the G domainof laminin α4) is evaluated on multiple occasions in multiple biologicalsamples from the patient, both to establish a measure of randomvariation and to show a trend in response to immunotherapy. The variousassessments of antibody binding to the biological samples are thencompared. If only two assessments are made, a direct comparison can bemade between the two assessments to determine whether antibody binding(i.e., presence of the LG4-5 modules of the G domain of laminin α4) hasincreased, decreased, or remained the same between the two assessments.If more than two measurements are made, the measurements can be analyzedas a time course starting before treatment with the therapeutic agentand proceeding through the course of therapy. In patients for whomantibody binding to biological samples has decreased (i.e., the presenceof the LG4-5 modules of the G domain of laminin α4 has decreased), itcan be concluded that the therapeutic agent was effective in treatingthe cancer in the patient. Preferably, the decrease in antibody bindingis statistically significant. Optionally, binding decreases by at least1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100%. Assessment of antibody binding can be made in conjunction withassessing other signs and symptoms of cancer.

The antibodies can also be used as research reagents for laboratoryresearch in detecting laminin α4, or more specifically, the LG4-5modules, or fragments thereof, of the G domain of laminin α4. In suchuses, antibodies can be labeled with fluorescent molecules, spin-labeledmolecules, enzymes, or radioisotopes, and can be provided in the form ofkit with all the necessary reagents to perform the assay for laminin α4,or more specifically, the LG4-5 modules of the G domain of laminin α4,or fragments thereof. The antibodies can also be used to purify lamininα4, laminins containing laminin α4, or binding partners of laminin α4,e.g., by affinity chromatography.

The antibodies can also be used for inhibiting cell adhesion in abiological sample. Preferably, the cell adhesion is dependent on lamininα4. For example, the cell adhesion is mediated by the LG4-5 modules ofthe G domain of laminin α4. The biological sample can comprises a tumor,cancer, or cells derived therefrom. Optionally, the tumor or cancer ismelanoma. An exemplary cell adhesion assay is described in the examples.In some instances, cell adhesion is inhibited by at least 10%, 20%, 25%,30%, 40%, 50%, or 75%, (e.g., 10%-75% or 30%-70%)

The antibodies can also be used for inhibiting binding of laminin α4 toa syndecan in a biological sample. Optionally, the syndecan issyndecan-1, syndecan-2, syndecan-3, or syndecan-4. Inhibition may bedemonstrated in a binding assay assessing the ability ofsyndecan-expressing cells to bind laminin α4 in the presence or absenceof the antibodies. Optionally, inhibition of a test antibody can bedemonstrated in comparison to an irrelevant control antibody not bindingto the LG4-5 modules of the G domain of laminin α4 or in comparison tovehicle lacking any antibody. For example, binding of laminin α4 to thesyndecan is inhibited by at least 10%, 20%, 25%, 30%, 40%, 50%, or 75%,(e.g., 10%-75% or 30%-70%).

The antibodies can also be used for inhibiting laminin-α4-induced pAktactivation in a biological sample. An exemplary assay is described inthe examples. In some methods, laminin-α4-induced pAkt activation isinhibited by at least 10%, 20%, 25%, 30%, 40%, 50%, or 75%, (e.g.,10%-75% or 30%-70%).

All patent filings, websites, other publications, accession numbers andthe like cited above or below are incorporated by reference in theirentirety for all purposes to the same extent as if each individual itemwere specifically and individually indicated to be so incorporated byreference. If different versions of a sequence are associated with anaccession number at different times, the version associated with theaccession number at the effective filing date of this application ismeant. The effective filing date means the earlier of the actual filingdate or filing date of a priority application referring to the accessionnumber if applicable. Likewise if different versions of a publication,website or the like are published at different times, the version mostrecently published at the effective filing date of the application ismeant unless otherwise indicated. Any feature, step, element,embodiment, or aspect of the invention can be used in combination withany other unless specifically indicated otherwise. Although the presentinvention has been described in some detail by way of illustration andexample for purposes of clarity and understanding, it will be apparentthat certain changes and modifications may be practiced within the scopeof the appended claims.

EXAMPLES Example 1. Identification of LG4-5-Domain-Specific Anti-LAMA4Monoclonal Antibodies

LAMA4 tumor expression has been reported to correlate with tumor grade,recurrence, and survival, while downregulation has been reported toinhibit tumor invasion in vitro and in vivo. See, e.g., Ljubimova etal., Cancer 101: 604-612 (2004) and Nagato et al., Int J Cancer 117:41-50 (2005). Interestingly, the LG4-5 modules of the G domain of LAMA4homologue LAMA3 have been reported to be exclusively found in squamouscell carcinomas (SCC) but absent/processed in normal healthy tissue.Furthermore, the LG4-5 modules of the G domain of LAMA3 have beenreported to be necessary for SCC tumor growth, as a polyclonal antibodyhas been reported to inhibit tumor growth in a SCC xenograft mousemodel. See, e.g., Tran et al., Cancer Res. 68: 2885-2894 (2008). It wasthus of great interest to determine (1) if LAMA4 is processed in asimilar fashion in tumors and (2) whether an LG4-5-specific anti-LAMA4antibody could be efficacious in LAMA4-positive tumors.

Monoclonal antibodies against the LG4-5 modules of the G domain of LAMA4were generated as described in Materials and Methods. The specificbinding between the monoclonal antibodies and the LG4-5 modules of the Gdomain of LAMA4 was confirmed by assessing the monoclonal antibodies'ability to stain LAMA4-fragment-displaying human embryonic kidney 293cells by flow cytometry. Fluorescent signal was assessed via flowcytometric analyses and plotted as mean fluorescence intensity (MFI) asshown in FIG. 1. A LG1-3-specific LAMA4 antibody was able to bind 293cells displaying LG1-5 and LG1-3, but not LG4-5 of the LAMA4 protein.Conversely, clones 6C12, 13G10, and 15F7 were able to specifically bind293 cells displaying LG1-5 and LG4-5, but not LG1-3.

Relative binding and on/off rates for the 15F7, 6C12, and 13G10antibodies were analyzed by ForteBio as shown in FIG. 2A-C,respectively. Antibody concentrations were kept constant at 100 nM, andthe concentration of LAMA4 was varied as indicated in FIG. 2A-C. Foreach concentration of LAMA4, two lines are presented in FIG. 2A-C: abolded line representing the raw data and a non-bolded line representingthe statistical fitting of the raw data. Detailed binding kineticparameters (association rate (1c), dissociation rate (k_(d)), andbinding affinity constant (K_(D))) were determined by Biacore for 13G10,15F7, and 6C12, as shown in Table 1. The 15F7 antibody displayed thehighest binding magnitude and slowest off rates in the ForteBio andBiacore assays.

TABLE 1 Biacore Assay Comparing Binding of 15F7, 13G10, and 6C12 toLAMA4 Human LAMA4 Murine LAMA4 Antibody k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D)(M) k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) 13G10 3.02 × 10⁵  7.4 × 10⁻³2.44 × 10⁻⁸ 3.78 × 10⁵ 4.49 × 10⁻³ 1.19 × 10⁻⁸ 15F7 1.14 × 10⁶ 7.81 ×10⁻⁴  6.84 × 10⁻¹⁰ 1.05 × 10⁶ 6.58 × 10⁻⁴ 6.27 10⁻¹⁰ 6C12 3.28 × 10⁵1.02 × 10⁻³ 3.11 × 10⁻⁹ 4.69 × 10⁵ 1.11 × 10⁻³ 2.38 × 10⁻⁹

The 6C12, 13G10, and 15F7 antibodies, along with mouse IgG control, weretested for their ability to bind LAMA4-displaying cells. To test LAMA4binding capacity, serially diluted antibodies were pre-incubated withLAMA4-displaying human 293 cells, followed by anti-human-650 secondaryantibody incubation as described in the Materials and Methods. Bindingcapacity was assessed by flow cytometry as shown in FIG. 3. 15F7 againdisplayed the highest binding magnitude.

Competition experiments were carried out to differentiate the 6C12,13G10, and 15F7 antibodies by epitope binding. Binding of the antibodiesto LAMA4-displaying 293 cells was assessed using decreasing ratios (5:1,1:1, and 1:5) of blocking antibody to binding antibody, with mouse IgG1being used as a negative control. Binding of the 6C12, 13G10, and 15F7antibodies was assessed by flow cytometery as shown in FIG. 4A-C,respectively. Fluorescent signal was assessed via flow cytometricanalyses and plotted as mean fluorescence intensity (MFI). All threeantibodies were able to compete with each other for LG4-5 binding, witheach being having higher blocking efficacy at the 5:1 ratio ((blockingantibody):(binding antibody)) and lower blocking efficacies as the ratiodecreases. These results indicate that these antibodies all bind similarepitopes on the LAMA4 protein.

Example 2. LG4-5-Domain-Specific Anti-LAMA4 Monoclonal AntibodyExclusively Stains Human and Mouse Melanoma Tissue

To determine whether the LG4-5 modules of the G domain of LAMA4 areexclusively found in tumor tissue, WM-266-4 human melanoma cells weretested for LAMA4 expression with polyclonal anti-LAMA4 antibodies.Staining was undertaken for both MCAM and LAMA4. MCAM-positive WM-266-4cells were positive for LAMA4 expression.

WM-266-4 tumors were implanted subcutaneously in nude mice and allowedto grow for five weeks. Following transcardiac perfusion with PBS,tumors and healthy tissue were frozen, sectioned, and stained with mouseIgG control, LG1-3-specific, and LG4-5-specific monoclonal antibodies6C12, 13G10, and 15F7 as described in Materials and Methods.LG1-3-specific and LG4-5-specific antibodies were both able to stainWM-266-4 subcutaneous tumors and vasculature.

Additional subcutaneous tumor experiments were performed using a mousemelanoma cell line, B16. B16 mouse melanoma cells were subcutaneouslyinjected or intravenously injected into mice and allowed to metastasizeto the lungs. Staining of B16 tumor tissue and healthy mouse brain,liver, kidney, and lung tissues was undertaken with a LG1-3-specificantibody, with mouse IgG1 used as a control. The LG1-3-specific antibodywas able to robustly stain all tissue types tested. Staining of B16tumor tissue and healthy mouse brain, liver, kidney, and lung tissueswas also undertaken with the LG4-5-specific antibodies, 6C12, 13G10, and15F7. In contrast to the LG1-3-specific antibody, these threeLG4-5-specific antibodies exclusively stained the B16 tumor vasculaturein the primary subcutaneous tumors and were not able to robustly stainany of the healthy tissues. Staining of a sample containing B16 lungmetastatic foci and healthy adjacent lung tissue was undertaken with theLG1-3-specific antibody, the LG4-5-specific antibodies (6C12, 13G10, and15F7), and a control mouse IgG1. The LG1-3-specific antibody was able torobustly stain both the metastatic foci and the healthy adjacent tissue.In contrast, the 6C12, 13G10, and 15F7 antibodies were not able to stainthe healthy adjacent lung tissue and exclusively stained B16 tumorvasculature in metastatic foci.

Samples of healthy human skin and skin melanoma from two distinctpatients were stained with a LG4-5-specific antibody, 15F7. Consistentwith the staining results described above, 15F7 was able to stain bothskin melanoma samples but was unable to stain the healthy skin tissue.

Other human tumor types were stained as well. As shown in FIG. 5A-C, aLG-4-5-specific antibody, 15F7, was used to stain tumor microarrayslides for human breast, colon, and lung tumors, respectively. Normaltissue controls are enclosed in the white rectangles. Consistent withthe staining of the melanomas described above, 15F7 preferentiallystained the majority of tumor patient samples in the tumor microarrayslides for human breast, colon, and lung cancers. These results areconsistent with a model whereby the LG4-5 modules of the G domain ofLAMA4 are exclusively found in various human and mouse tumor types,indicating that the LG4-5 modules of the G domain of LAMA4 can be atumor-specific marker for diagnostics and targeted tumor therapeutics.

Example 3. LG4-5-Domain-Specific Anti-LAMA4 Monoclonal Antibody InhibitsHuman Melanoma Cell Adhesion and Drives Antibody-Drug Conjugate CellToxicity

To determine the functional consequences of targeting the LG4-5 modulesof the G domain of LAMA4 with anti-LG4-5 antibodies, recombinantLG4-5-coated ELISA plates were incubated with 20 ug/ml 15F7, 6C12, and13G10 (or mouse IgG1 control) and were then assayed for their ability tobind human melanoma cell line WM-266-4 as described in Materials andMethods. Buffer was used as a negative control and heparin was used as apositive control. A sample with no cells was used as an additionalnegative control. The results of the cell adhesion assay are shown inFIG. 6. The results are presented in arbitrary units (A.U.) on they-axis. Although mouse IgG1 control failed to block LAMA4-mediated celladhesion, all three anti-LG4-5 antibodies were able to inhibitLAMA4-mediated human melanoma cell adhesion by approximately 35% orgreater, indicating that anti-LG4-5 antibodies can block cell adhesionevents necessary for tumor cell adhesion, proliferation, and metastasis.

Because the LG4-5 domain of LAMA4 is highly enriched in various humantumor tissues when compared to healthy human tissue, we testedanti-LG4-5 antibodies as candidates for antibody-drug conjugatetargeting strategies. As shown in FIG. 7, LAMA4+WM-266-4 cells wereincubated with the 15F7 anti-LG4-5 antibody or a mouse isotype controlalong with ribosomal toxin saporin-conjugated anti-mouse secondary.Although LAMA4 LG4-5 is a soluble extracellular matrix protein, 15F7 wasable to strongly mediate saporin-mediated cell toxicity.

These data indicate that anti-LG4-5 antibodies block WM-266-4 humanmelanoma cell adhesion and are strong candidates for antibody-drugconjugate therapeutic approaches. Targeting LG4-5 could be efficaciousin slowing tumor growth and metastasis.

Example 4. Design of Humanized 15F7 Antibodies

The starting point or donor antibody for humanization was the mouseantibody 15F7. The heavy chain variable amino acid sequence of maturem15F7 is provided as SEQ ID NO:16. The light chain variable amino acidsequence of mature m15F7 is provided as SEQ ID NO:17. The heavy chainCDR1, CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOS:20,21, and 22, respectively (as defined by Kabat). The light chain CDR1,CDR2, and CDR3 amino acid sequences are provided as SEQ ID NOS:23, 24,and 25, respectively (as defined by Kabat). Kabat numbering is usedthroughout in this Example.

The variable kappa (Vk) of m15F7 belongs to mouse Kabat subgroup 5,which corresponds to human Kabat subgroup 1. The variable heavy (Vh) ofm15F7 belongs to mouse Kabat subgroup 5a, which corresponds to Kabatsubgroup 1. See Kabat et al. Sequences of Proteins of ImmunologicalInterest, Fifth Edition. NIH Publication No. 91-3242, 1991. The11-residue CDR-L1 belongs to canonical class 1, the 7-residue CDR-L2belongs to canonical classl, and the 9-residue CDR-L3 belongs tocanonical class 1 in Vk. See Martin & Thornton, J. Mol. Biol.263:800-15, 1996. The 5-residue CDR-H1 (as defined by Kabat) belongs tocanonical class 1, and the 17-residue CDR-H2 belongs to canonicalclass 1. See Martin & Thornton, J Mol. Biol. 263:800-15, 1996. TheCDR-H3 has no canonical classes, but the 10-residue loop probably has akinked base according to the rules of Shirai et al., FEBS Lett.455:188-97 (1999).

The residues at the interface between the Vk and Vh domains are usual.

A search was made over the protein sequences in the PDB database(Deshpande et al., Nucleic Acids Res. 33: D233-7, 2005) to findstructures which would provide a rough structural model of 15F7. Thecrystal structure of the antibody against monocyte chemoattractantproteins (MCPs) was used for Vk structure. It retains the same canonicalstructure for the loop as 15F7 (pdb code 2BDN, resolution 2.53 A). Theheavy chain of the antibody against LeuT mutants (pdb code 3TT1,resolution 3.1 A) was used for Vh structure. It contains the samecanonical structures for CDR-H1 and CDR-H2 as that of 15F7 VH, and alsothe same length CDR-H3 with a kinked base. BioLuminate was used to modela rough structure of 15F7 Fv.

A search of the non-redundant protein sequence database from NCBI withCDR“X”ed 15F7 Fv allowed selection of suitable human frameworks intowhich to graft the murine CDRs. For Vh, two human Ig heavy chains,ACF36857.1 and BAC01530.1 (SEQ ID NOS:96 and 97, respectively) werechosen. They share the canonical form of 15F7 CDR-H1 and H2, and H3 ofBAC01530.1 is 10 residues long with a predicted kinked base. For Vk, twohuman kappa light chains, having NCBI accession codes AAY33350.1 andBAC01583.1 (SEQ ID NOS:53 and 98, respectively), were chosen. They havethe same canonical classes for CDR-L1, L2, and L3 as that for theparental Vk. Humanized 15F7 heavy and light chain variable regionsequences having no backmutations or other mutations are provided as SEQID NOS:55 and 56.

Two humanized heavy chain variable region variants and two humanizedlight chain variable region variants were constructed containingdifferent permutations of substitutions (Hu15F7VHv1-2 (SEQ ID NOS:57 and58, respectively) and Hu15F7VLv1-2 (SEQ ID NOS:59 and 60, respectively))(FIGS. 12A-D, FIGS. 13A-D). The exemplary humanized Vh and Vk designs,with backmutations and other mutations based on selected humanframeworks, are shown in FIGS. 12A-D and FIGS. 13A-D, respectively. Thegray-shaded areas in the first column in FIGS. 12A-D and FIGS. 13A-Dindicate the CDRs as defined by Chothia, and the gray-shaded areas inthe remaining columns in FIGS. 12A-D and FIGS. 13A-D indicate the CDRsas defined by Kabat. SEQ ID NOS:57-60 contain backmutations and othermutations as shown in Table 2. The amino acids at positions H1, H20,H27, H30, H38, H40, H41, H48, H66, H67, H69, H75, H82A, H91, L8, L42,L45, L49, L69, and L80 in Hu15F7VHv1-2 and Hu15F7VLv1-2 are listed inTable 3. The amino acids at position L104 in Hu15F7VLv1-2 are alsolisted in Table 3.

TABLE 2 V_(H), V_(L) Backmutations and Other Mutations V_(L) VariantV_(L) Exon Acceptor Sequence Donor Framework Residues Hu15F7VLv1 NCBIaccession codes AAY33350.1 L8, L42, L45, L49, L69, L80 (SEQ ID NO: 59)and BAC01583.1 (SEQ ID NOS: 53 and 98) Hu15F7VLv2 NCBI accession codesAAY33350.1 L42, L49, L69, L104 (SEQ ID NO: 60) and BAC01583.1 (SEQ IDNOS: 53 and 98) Hu15F7VHv1 NCBI accession codes ACF36857.1 H1, H20, H27,H30, H38, H40, (SEQ ID NO: 57) and BAC01530.1 H41, H48, H66, H67, H69,H75, (SEQ ID NOS: 96 and 97) H82A, H91 Hu15F7VHv2 NCBI accession codesACF36857.1 H1, H20, H27, H30, H38, H40, (SEQ ID NO: 58) and BAC01530.1H48, H66, H67, H75, H82A, H91 (SEQ ID NOS: 96 and 97)

TABLE 3 Kabat Numbering of Framework Residues for Backmutations andOther Mutations in Humanized 15F7 Antibodies AAY33350.1 BAC01583.1ACF36857.1 BAC01530.1 Mouse Hu15F Hu15F Hu15F Hu15F Residue light chainlight chain heavy chain heavy chain 15F7 7VL1 7VL2 7VH1 7VH2 L8 P P — —S S P — — L42 K K — — N N N — — L45 K K — — R R K — — L49 Y Y — — S S S— — L69 T T — — K K K — — L80 P P — — T T P — — L104 L L — — L L V — —H1 — — Q Q Q — — E E H20 — — V V L — — L L H27 — — G G Y — — Y Y H30 — —S S T — — T T H38 — — R R K — — K K H40 — — A A R — — R R H41 — — P P A— — A P H48 — — M M I — — I I H66 — — R R K — — K K H67 — — V V A — — AA H69 — — I I L — — L I H75 — — T T S — — S S H82A — — S S R — — R R H91— — Y Y F — — F F

The rationales for selection of the above positions in the light chainvariable region as candidates for substitution are as follows.

P8S: P is more frequent than S in the human IgG framework, but becauseproline cis-trans isomerization affects protein folding, P was tried inone version and S in the other version.

K42N: N contacts interface residue F91 in VH and is therefore criticalfor maintaining antibody structure.

K45R: R and K have similar frequency in the human IgG framework, so Rwas tried in one version and K in the other version.

Y49S: S contacts LCDR2 and is critical.

T69K: K contacts LCDR1 and is critical.

P80T: P is more frequent than T in the human IgG framework, but becauseproline cis-trans isomerization affects protein folding, P was tried inone version and T was tried in the other version.

L104V: L was tried in one version, and V was tried in the other version.

The rationales for selection of the above positions in the heavy chainvariable region as candidates for substitution are as follows.

Q1E: This is a mutation but not a backmutation. Glutamate (E) conversionto pyroglutamate (pE) occurs more slowly than from glutamine (Q).Because of the loss of a primary amine in the glutamine to pEconversion, antibodies become more acidic. Incomplete conversionproduces heterogeneity in the antibody that can be observed as multiplepeaks using charge-based analytical methods. Heterogeneity differencesmay indicate a lack of process control.

V20L: L is more frequent than V in the human IgG framework.

G27Y: This residue is within HCDR1 as defined by Chothia, so Y was usedto maintain binding ability.

S30T: This residue is within HCDR1 as defined by Chothia, so T was usedto maintain binding ability.

R38K: K contacts two interface residues, L45 and W47, in VH and istherefore critical.

A40R: R is located at the end of the KQRAGQG (VH amino acids 38-44)loop, which supports the two interface residues, V37 and L45 in VH. Itis important for maintaining antibody folding.

P41A: P is more frequent than A in the human IgG framework, butintroduction of P into the framework may cause some conformationalchanges, so P was tried in one version and A was tried in the otherversion.

M48I: I contacts HCDR2 and interface residues V37 and L45 in VH.

R66K: K contacts HCDR2.

V67A: A contacts HCDR2.

I69L: I is more frequent than L in the human IgG framework, but Lcontacts HCDR1 and HCDR2, so I was tried in one version and L was triedin the other version.

T75S: Both T and S are frequent in the human framework.

S(82A)R: R contacts HCDR2 and is critical.

Y91F: F is an interface residue and is important to support antibodyfolding.

The two humanized light chain variable region variants and two humanizedheavy chain variable region variants are as follows:

Hu15F7VL version 1 (P8S, K42N, K45R, Y49S, T69K, and P80T backmutationsin lowercase):

(SEQ ID NO: 59) DIQMTQSsSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGnAPrLLIsGATSLETGVPSRFSGSGSGkDYTLTISSLQtEDFATYYCQQYWSIPYTF GGGTKLEIKR.

Hu15F7VL version 2 (K42N, Y49S, and T69K backmutations and L104Vmutation in lowercase):

(SEQ ID NO: 60) DIQMTQSPSSLSASVGDRVTITCKASEDIYNRLAWYQQKPGnAPKLLIsGATSLETGVPSRFSGSGSGkDFTLTISSLQPEDFATYYCQQYWSIPYTF GGGTKvEIKR.

Hu15F7VH version 1 (Q1E mutation and V20L, G27Y, S30T, R38K, A40R, P41A,M48I, R66K, V67A, I69L, T75S, S(82A)R, and Y91F backmutations inlowercase):

(SEQ ID NO: 57) eVQLQQSGAEVKKPGSSVKlSCKASGyTFtSYGLSWVkQraGQGLEWiGEIFPRSGNTYYNEKFKGkaTlTADKSsSTAYMELrSLRSEDTAVYfCAR GVRSPGAMDYWGQGTLVTVSS.

Hu15F7VH version 2 (Q1E mutation and V20L, G27Y, S30T, R38K, A40R, M48I,R66K, V67A, T75S, S(82A)R, and Y91F backmutations in lowercase):

(SEQ ID NO: 58) eVQLQQSGAEVKKPGSSVKlSCKASGyTFtSYGLSWVkQrPGQGLEWiGEIFPRSGNTYYNEKFKGkaTITADKSsSTAYMELISLRSEDTAVYfCAR GVRSPGAMDYWGQGTLVTVSS.

Example 5. Binding Kinetic Analysis of Humanized 15F7 Antibodies

Binding kinetics of humanized 15F7 antibodies comprising a heavy chainselected from Hu15F7VHv1-2 (H1 and H2) and a light chain selected fromHu15F7VLv1-2 (L1 and L2) were characterized.

Chimeric 15F7, H1L1, H1L2, H2L1, H2L2, and buffer alone were tested fortheir ability to bind to LAMA4-fragment-displaying cells. To test LAMA4binding capacity, serially diluted antibodies were pre-incubated withhuman 293 cells displaying LG4-5, followed by anti-human-650 secondaryantibody incubation as described in the Materials and Methods.Fluorescent signal was assessed via flow cytometric analyses and plottedas mean fluorescence intensity (MFI) as shown in FIG. 8. The seriallydiluted H1L1, H1L2, H2L1, and H2L2 antibodies each showed bindingcapacity that is comparable to chimeric 15F7.

The specific binding of the chimeric 15F7, H1L1, H1L2, H2L1, and H2L2antibodies to the LG4-5 modules of the G domain of LAMA4 was tested byassessing the antibodies' ability to stain LAMA4-fragment-displaying 293cells by flow cytometry, as shown in FIG. 9. Each of the antibodies wasable to specifically bind 293 cells displaying LG1-5 and LG4-5, but notLG1-3.

Relative binding and on/off rates were analyzed by ForteBio, as shown inFIGS. 10A and 10B. In FIG. 10A, the anti-His sensor was loaded with 10ug/ml of purified His-LAMA4 followed by loading of 5 ug/ml of m15F7,chimeric 15F7, H1L1, H1L2, H2L1, and H2L2. Association and dissociationwere analyzed. In FIG. 10B, the goat anti-human Fc sensor was loadedwith m15F7, chimeric 15F7, H1L1, H1L2, H2L1, and H2L2 as indicated at 5ug/ml followed by loading of 10 ug/ml of His-LAMA4. Association anddissociation were analyzed. Relative binding and on/off rates werecomparable among all antibodies tested, with H1L1 and H2L1 displayingrelative binding that was the same or higher than that for chimeric 15F7and m15F7.

Biacore full binding kinetic analysis of antibodies was then carriedout. SPR analysis was performed as described in the Materials andMethods. Detailed binding kinetic parameters (association rate(k_(assoc)), dissociation rate (k_(dissoc)), and binding affinityconstant (K_(d))) were determined for chimeric 15F7, humanized H1L1, andhumanized H2L1. Binding kinetic parameters for the humanized 15F7variants H1L1 and H2L1 were comparable to those for chimeric 15F7 (seeTable 4).

TABLE 4 Biacore Assay Comparing Binding of HU15F7 Variants and Chimeric15F7 to LAMA4 Antibody k_(assoc) (M⁻¹s⁻¹) k_(dissoc) (s⁻¹) K_(d) (M)Chimeric 1.50 × 10⁶ 1.51 × 10⁻⁴ 1.0 × 10⁻¹⁰ H1L1 1.50 × 10⁶ 1.83 × 10⁻⁴1.2 × 10⁻¹⁰ H2L1 1.56 × 10⁶ 2.05 × 10⁻⁴ 1.3 × 10⁻¹⁰

Example 6. Anti-Laminin Antibodies Inhibit Laminin-411-Induced pAktActivation

WM266.4 human tumor melanoma cells were serum-starved for 24 h and thenresuspended into serum-free cell culture media with 10 ug/ml laminin 411(LAMA4 in complex with gamma1 and beta1 chains) and 20 ug/ml 15F7 ormIgG1 control antibody for 30 minutes. BSA protein was used as a controlfor laminin 411. Cells were then spun down and lysed for immunoblotanalyses. pAkt and total Akt levels were assessed by immunoblot. Ratiosof these levels (pAkt/Akt) are shown in FIGS. 11A & B. Each condition(mIgG1+laminin 411; 15F7+laminin 411; and mIgG1+BSA) was tested intriplicate. FIG. 11A shows the results for each individual sample, andFIG. 11B shows the averages and standard errors for each condition. Asshown in FIG. 11B, laminin 411 induced pAkt signaling (i.e., higherpAkt/Akt ratio) compared to BSA control, and 15F7 partially blockedlaminin-411-induced pAkt activation (˜50% inhibition).

Example 7. Effects of Laminin 411 and Anti-Laminin Antibodies on NotchSignaling

Because Notch ligand D114 transcription/translation requires integrinligation and subsequent phospho-Akt signaling, anti-LAMA4 antibodies aretested for effects on Notch signaling. HUVEC, WM266.4, and RAW cells areresuspended in cell culture media with 10 ug/ml laminin-411 (LAMA4 incomplex with gamma1 and beta1 chains) and 20 ug/ml 15F7 or isotypecontrol antibody for 24 hrs. BSA protein is used as a control forlaminin 411. Cells are spun down and lysed for immunoblot analyses forcleaved/activated Notch1, D114, MCAM, actin, pAkt, and Akt. In addition,qPCR analysis for Hey1, MCP-1 (monocyte chemoattractant ininflammation), MCAM, LAMA4, and GAPDH is undertaken.

Example 8. Effects of Anti-Laminin Antibodies in In Vivo Obesity Models

Because Akt signaling is important for Notch signaling, and Notchsignaling encourages growth of adipocytes, antibodies against LAMA4 aretested in in vivo obesity models for effects on weight gain/loss andadipocyte metabolism and lipolysis. High-fat diet (HFD)-driven weightgain in mice is assessed in response to anti-laminin 411 antibodies.Wild-type C57BL/6 mice are fed a high-fat diet (e.g., rodent diet with45% kcal % fat, such as product #D12451 from Research Diets, Inc.) adlibitum. Two experimental groups are tested: (1) mice treated withcontrol Ig; and (2) mice treated with 15F7. There are ten mice in eachgroup, and each mouse is treated with 10 mg/kg/week antibody for threeto four months. Weight measurements are taken every two to four weeks.

To assess localization of LAMA4 to adipose tissue, anti-LAMA4 antibody(compared to isotype control antibody) is intravenously administered tomice. Staining is then undertaken to assess localization to adiposetissue.

Example 9. Materials and Methods LAMA4 Fragment Purification

His-tagged LAMA4 G-domain fragments were cloned by standard proceduresand transiently expressed in 293 cells. Protein was purified using anickel-NTA column.

LAMA4 Knockout Mouse

Lama4 null mice originally obtained from Dr. Karl Tryggvason (KarolinskaUniversity).

Generation of Recombinant MCAM-Fc Protein

MCAM-Fc was generated in house by fusing the extracellular domain ofhuman or mouse MCAM to human IgG1 and produced/purified in CHO cellsusing standard techniques.

Antibody Generation

Recombinant mouse laminin 4 (Lama4) obtained from R&D Systems and 10week old Lama4 null mice originally obtained from Dr. Karl Tryggvason(Karolinska University) were used to develop the antibodies. Purifiedlaminin α4 (LAMA4) was suspended in RIBI adjuvant at 10 ug LAMA4/25 uladjuvant. Mice were anesthetized with isoflurane and 3 mice wereimmunized into each rear footpad or rear hock with 5 ug Lama4 in RIBIadjuvant while two mice were immunized with 12.5 ug Lama4 in RIBIadjuvant into each rear footpad or rear hock with a 27 gauge needle.Mice were injected following the above procedure on days 0, 4, 12, 16and 20. On day 24 animals are euthanized and the popiteal and inguinallymph nodes are removed in a sterile hood. The nodes are dissociated andfused with SP2/0 using a modification of the Kohler and Milsteinprotocol that incorporates Electrofusion instead of PEG fusion. Fusedcells are plated into 96 well plates and allowed to grow.

When cells reach half to three quarters confluence screening begins.Briefly, Costar RIA/EIA plates were coated with rabbit ant-His tag(Anaspec #29673) at 1 ug/mL, 50 uL/well, in PBS for 1 hour. Plates werethen blocked with 250 ul/well of 1% BSA/PBS for 15 minutes and thenremoved. His-tagged Lama4 was added to the plates at 0.25 ug/mL, 50uL/well for 1 hour, and then washed 2×. 75 uL of supernatant from fusionplates was added and incubated for 1 hour, plates were washed 2×.Goat-anti-mouse (Jackson #115-035-164) was added at 1:2000 dilution in0.5% BSA/PBS/TBST for 1 hour, then washed 5×. Plates were developed with50 ul/well TMB (SurModics #TMBW24) for 5 minutes, and stopped with 15 uL2N H2SO4, and read at 450 nm. Wells with OD greater than 1.0 wereselected for additional screening. Cells from wells found positive bythe ELISA were grown up and frozen. Supernatants were provided for theadditional screening described below. Cells from wells meeting certaincriteria described below were cloned using the Clonepix FL and screenedusing setting recommended by the company to find single cell clones.These were expanded and the antibody purified from supernatants.

LAMA4 pDisplay Flow Cytometric Binding Assay

Human LAMA4 G-domains 1-5 and variants were cloned into pDisplayexpression construct (Life Technologies) and transiently transfectedinto 293 cells using standard procedures. Anti-LAMA4 antibodies wereincubated with cells for 30 min at 4° C. and followed by anti-mouse-650for 30 minutes at 4° C. Cells were analyzed for anti-laminin binding byflow cytometry using standard procedures.

WM-266-4 Cell Transfection and Staining

Cultured WM-266-4 cells were transfected with full-length human MCAM-GFPfusion constructs and stained with anti-LAMA4 antibodies using standardprocedures.

WM-266-4 Human Melanoma Subcutaneous Tumor Tissue

Cells were cultured and 5×10⁵ cells per animal were subcutaneouslyadministered above the shoulder in nude mice. After five weeks, animalswere transcardially perfused with PBS and tumors were excised and snapfrozen.

B16 Mouse Melanoma Subcutaneous Tumor Tissue

Cells were cultured and 5×10⁵ cells per animal were subcutaneouslyadministered above the shoulder in c57b16 mice. After five weeks,animals were transcardially perfused with PBS and tumors were excisedand snap frozen.

B16 Mouse Melanoma Lung Metastasis Lung Tissue

Cells were cultured and 5×10⁵ cells per animal were intravenouslyinjected into nude/beige mice from Charles River (offsite at CaliperLifeSciences). After three weeks, animals were transcardially perfusedwith PBS, and lungs were excised and snap frozen.

Fluorescence Microscopy/Standard Immunofluorescent Methods

Mouse tissue was snap frozen in OCT and sectioned at 10 uM. Sectionswere fixed in cold acetone and stained with anti-LAMA4 antibody (R&Dsystems).

Human Melanoma Cell Adhesion Assay

Recombinant 10 ug/ml mLAMA4 (R&D systems), was used to coat 96-wellplates overnight at 4° C. Following PBS washing steps, wells wereblocked with 1% BSA/MEM for 1 hr at room temperature. 20 ug/mlanti-LAMA4 antibodies in 0.1% BSA/MEM were added to plates for 1 hour atroom temperature. WM-266-4 cells were resuspended with EDTA, wash andresuspended at 300,000 cells/ml in 0.1%/MEM, followed by 10 minutes inthe tissue culture incubator at 37° C. with the tube cap off. Followingtwo washes with FACS buffer (1% FBS in PBS), cells were resuspended with650-conjugated anti-pan-laminin antibody (1:1000; Novus Biologicals) andincubated for 20 min at 4° C., and washed again. Without removingantibody solutions, add cell suspension to well and incubate uncoveredin tissue culture incubator for 1.5 hrs. Following a PBS wash step,cells were stained/fixed with glutaraldehyde/crystal violet solutionprior to plate reader analysis at 570 nm.

Generation of Fab Fragments

Fab fragments of all antibodies were generated using the Fab MicroPreparation kit following manufacturer's directions (Pierce). Removal ofliberated Fc and verification of intact final product were monitored bySDS-PAGE, and concentration was determined using the bicinchoninic acidassay (Pierce).

SPR Measurements of Affinity

SPR analysis was performed using a Biacore T200 to compare the bindingof the different laminin antibodies. For Fab preparations, anti-6×Hisantibody (GE Life Sciences) was immobilized on sensor chip C1 via aminecoupling, and human His-laminin-α4, mouse His-laminin-α4 (both from R &D Systems), and an unrelated 6×His-tagged protein (as a reactioncontrol) were captured at a level to ensure maximum binding of 25 RU.Various concentrations of Fab preparations ranging from 300-0.41 nM werepassed over the captured ligands in parallel at a flow rate of 50 ul/minin running buffer (EMS+0.05% P-20, 1 mg/mL BSA), for 240 s associationand varying durations of dissociation. Data were double-referenced toboth an irrelevant sensor not containing His-tagged ligand, and 0 nManalyte concentration to account for the dissociation of ligand from thecapture moiety. Data was then analyzed using either a heterogeneousligand model or a global 1:1 fit.

For whole IgG, anti-mouse antibody was immobilized on sensor chip C1(lacking dextran chains) via amine coupling, and laminin mAbs werecaptured to a level to ensure a maximum binding of analyte of 50 RU.Various concentrations of analyte (recombinant human or mouseHis-laminin-α4 fragment starting at Q826 (R&D Systems)) ranging from31.25 nM to 0.122 nM were passed over the captured ligand at 30 ul/minin running buffer (EMS+0.05% P-20, 1 mg/mL BSA, except where described)for 180 s association/900 s dissociation. Data were double-referenced toboth an irrelevant sensor not containing His-tagged ligand, and 0 nManalyte concentration to account for the dissociation of ligand from thecapture moiety. Where possible, data were analyzed using a global 1:1fit. If kinetic sensorgram curvature did not allow proper model-fitting,a steady-state approximation was performed and reported.

Saporin-Mediated WM-266-4 Cell Toxicity Assay

Per manufacturer's instructions (Advanced Targeting Systems).

Human Melanoma Tissue and Tumor Microarray Slides

Unfixed healthy and melanoma human skin slides were obtained fromOrigene. Acetone-fixed tumor microarray (TMA) slides were obtained fromBiochain.

1-57. (canceled)
 58. A method of treating or effecting prophylaxis of acancer in a patient having or at risk for the cancer, the methodcomprising administering to the patient an effective regime of anantibody that specifically binds to an epitope within the LG4-5 modulesof the G domain of laminin α4.
 59. The method of claim 58, wherein thepatient has a cancer, and the cancer is melanoma, breast cancer, lungcancer, or colorectal cancer. 60-74. (canceled)
 75. A method of treatingor effecting prophylaxis of a disease in which the LG4-5 modules of theG domain of laminin α4 contribute to progression of the disease, themethod comprising administering to a patient having or at risk of thedisease an effective regime of an antibody that specifically binds to anepitope within the LG4-5 modules of the G domain of laminin α4. 76-83.(canceled)
 84. A method of treating or effecting prophylaxis of anautoimmune disease in a patient having or at risk for the autoimmunedisease, the method comprising administering to the patient an effectiveregime of an antibody that specifically binds to an epitope within theLG4-5 modules of the G domain of laminin α4.
 85. (canceled)
 86. A methodof inhibiting angiogenesis in a patient, the method comprisingadministering to the patient an effective regime of an antibody thatspecifically binds to an epitope within the LG4-5 modules of the Gdomain of laminin α4. 87-93. (canceled)
 94. A method of treating oreffecting prophylaxis of obesity or an obesity-related disease in apatient having or at risk for obesity or the obesity-related disease,the method comprising administering to the patient an effective regimeof an antibody that specifically binds to an epitope within the LG4-5modules of the G domain of laminin α4.
 95. (canceled)
 96. The method ofclaim 58, wherein the antibody comprises three light chain CDRs andthree heavy chain CDRs from the heavy and light chain variable regionsof 15F7 (SEQ ID NOS:16 and 17, respectively), 6C12 (SEQ ID NOS:26 and27, respectively), or 13G10 (SEQ ID NOS:36/37 and 38, respectively). 97.The method of claim 58, wherein the antibody comprises three light chainCDRs and three heavy chain CDRs from the heavy and light chain variableregions of 15F7 (SEQ ID NOS:16 and 17, respectively), 6C12 (SEQ IDNOS:26 and 27, respectively), or 13G10 (SEQ ID NOS:36/37 and 38,respectively).
 98. The method of claim 74, wherein the antibodycomprises three light chain CDRs and three heavy chain CDRs from theheavy and light chain variable regions of 15F7 (SEQ ID NOS:16 and 17,respectively), 6C12 (SEQ ID NOS:26 and 27, respectively), or 13G10 (SEQID NOS:36/37 and 38, respectively).
 99. The method of claim 84, whereinthe antibody comprises three light chain CDRs and three heavy chain CDRsfrom the heavy and light chain variable regions of 15F7 (SEQ ID NOS:16and 17, respectively), 6C12 (SEQ ID NOS:26 and 27, respectively), or13G10 (SEQ ID NOS:36/37 and 38, respectively).
 100. The method of claim86, wherein the antibody comprises three light chain CDRs and threeheavy chain CDRs from the heavy and light chain variable regions of 15F7(SEQ ID NOS:16 and 17, respectively), 6C12 (SEQ ID NOS:26 and 27,respectively), or 13G10 (SEQ ID NOS:36/37 and 38, respectively). 101.The method of claim 94, wherein the antibody comprises three light chainCDRs and three heavy chain CDRs from the heavy and light chain variableregions of 15F7 (SEQ ID NOS:16 and 17, respectively), 6C12 (SEQ IDNOS:26 and 27, respectively), or 13G10 (SEQ ID NOS:36/37 and 38,respectively).